Engineered biocatalysts and methods for synthesizing chiral amines

ABSTRACT

The present disclosure provides engineered transaminase polypeptides for the production of amines, polynucleotides encoding the engineered transaminases, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases to prepare compounds useful in the production of active pharmaceutical agents.

The present application is a Divisional of U.S. patent application Ser.No. 15/869,455, filed on Jan. 12, 2018, which is a Divisional of U.S.patent application Ser. No. 14/652,887, filed on Jun. 17, 2015, now U.S.Pat. No. 9,902,943, which is a national stage application filed under 35USC § 371, which claims priority to international application to PCTInternational Application No. PCT/US2013/075294, filed Dec. 16, 2013,which claims priority to US Provisional Appin. Ser. No. 61/745,219,filed Dec. 21, 2012, all of which are incorporated for all purposes intheir entireties.

1. TECHNICAL FIELD

The disclosure relates to transaminase biocatalysts and processes usingthe biocatalysts for the preparation of chiral amines.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-126USP1_ST25.txt”, a creation date of Dec. 21, 2012,and a size of 606,099 kilobytes. The Sequence Listing filed via EFS-Webis part of the specification and is incorporated in its entirety byreference herein.

3. BACKGROUND

Transaminases (E.C. 2.6.1) catalyze the transfer of an amino group, apair of electrons, and a proton from a primary amine of an amino donorsubstrate to the carbonyl group of an amino acceptor molecule as shownin Scheme 1.

An amino acceptor compound (I) (which is the precursor of the desiredchiral amine product (III)) is reacted with an amino donor compound(II). The transaminase catalyzes the transfer of the amine group of theamino donor (II) to the keto group of the amino acceptor (I). Thereaction results in the desired chiral amine product compound (III) anda new amino acceptor compound (IV) with a ketone group as a by-product.

Wild-type transaminases having the ability to catalyze a reaction ofScheme 1 have been isolated from various microorganisms, including, butnot limited to, Alcaligenes denitrificans, Bordetella bronchiseptica,Bordetella parapertussis, Brucella melitensis, Burkholderia malle,Burkholderia pseudomallei, Chromobacterium violaceum, Oceanicolagranulosus HTCC2516, Oceanobacter sp. RED65, Oceanospirillum sp. MED92,Pseudomonas putida, Ralstonia solanacearum, Rhizobium meliloti,Rhizobium sp. (strain NGR234), Bacillus thuringensis, Klebsiellapneumonia, and Vibrio fluvialis (see e.g., Shin et al., 2001, Biosci.Biotechnol, Biochem. 65:1782-1788). Several of these wild-typetransaminase genes and encoded polypeptides have been sequenced,including e.g., Ralstonia solanacearum (Genbank Acc. No. YP_002257813.1,GI:207739420), Burkholderia pseudomallei 1710b (Genbank Acc. No.ABA47738.1, GI:76578263), Bordetella petrii (Genbank Acc. No.AM902716.1, GI:163258032), and Vibrio fluvialis (Genbank Acc. No.AEA39183.1, GI: 327207066). Two wild-type transaminases of classes EC2.6.1.18 and EC 2.6.1-19, have been crystallized and structurallycharacterized (see e.g., Yonaha et al., 1983, Agric. Biol. Chem. 47(10):2257-2265).

The wild-type transaminase from from Vibrio fluvialis JS17 is an ω-aminoacid:pyruvate transaminase (E.C. 2.6.1.18) that usespyridoxal-5′-phosphate as cofactor to catalyze the reaction of Scheme 2.

This wild-type transaminase from Vibrio fluvialis also has been reportedto show catalytic activity toward aliphatic amino donors that do nothave a carboxyl group.

Chiral amine compounds are frequently used in the pharmaceutical,agrochemical and chemical industries as intermediates or synthons forthe preparation of various pharmaceuticals, such as cephalosporine orpyrrolidine derivatives. A great number of these industrial applicationsof chiral amine compounds involve using only one particular opticallyactive form, e.g., only the (R) or the (S) enantiomer is physiologicallyactive. Transaminases have potential industrial use for thestereoselective synthesis of optically pure chiral amine compounds, suchas in the enantiomeric enrichment of amino acids (see e.g., Shin et al.,2001, Biosci. Biotechnol. Biochem. 65:1782-1788; Iwasaki et al., 2003,Biotech. Lett. 25:1843-1846; Iwasaki et al., 2004, Appl. Microb.Biotech. 69:499-505, Yun et al., 2004, Appl. Environ. Microbiol.70:2529-2534; and Hwang et al., 2004, Enzyme Microbiol. Technol.34:429-426).

Other examples of the use of transaminases include the preparation ofintermediates and precursors of pregabalin (e.g., WO 2008/127646); theenzymatic transamination of cyclopamine analogs (e.g., WO 2011/017551);the stereospecific synthesis and enantiomeric enrichment of β-aminoacids (e.g., WO 2005/005633); the enantiomeric enrichment of amines(e.g., U.S. Pat. Nos. 4,950,606; 5,300,437; and 5,169,780); and theproduction of amino acids and derivatives (e.g., U.S. Pat. Nos.5,316,943; 4,518,692; 4,826,766; 6,197,558; and 4,600,692).

However, transaminases used to catalyze reactions for the preparation ofchiral amine compounds can have properties that are undesirable forcommercial applications, such as instability to industrially usefulprocess conditions (e.g., solvent, temperature) and narrow substraterecognition. Thus, there is a need for other types of transaminasebiocatalysts that can be used in industrial processes for preparingchiral amines compounds in an optically active form.

4. SUMMARY

The present disclosure provides engineered polypeptides havingtransaminase activity, polynucleotides encoding the polypeptides,methods of the making the polypeptides, and methods of using thepolypeptides for the biocatalytic conversion of ketone substrates toamine products. The polypeptides having transaminase activity of thepresent disclosure have been engineered to have one or more residuedifferences as compared to a previously engineered transaminasepolypeptide (of amino acid sequence SEQ ID NO: 2) with enhanced solventand thermal stability relative to the wild-type transaminase of Vibriofluvialis. The amino residue differences are located at residuepositions affecting various enzyme properties, including among others,activity, stereoselectivity, stability, expression, and producttolerance. In particular, the engineered transaminase polypeptides ofthe present disclosure have been engineered for efficient conversion ofan exemplary large cyclopamine analog ketone compound of compound (2) toits corresponding chiral amine product compound of compound (1) as shownin Scheme 3.

The evolved structural features of the engineered transaminasepolypeptides of the present disclosure also allow for the conversion ofa range of large ketone substrate compounds (other than the compound(2)), such as cyclopamine analogs, veratramine analogs, and steroidanalogs, of Formula (II) to their corresponding chiral amine productcompounds of Formula (I) as shown in Scheme 4.

wherein rings A-D of the compounds can be substituted as follows:

Ring A is a 6-membered carbocyclic ring, optionally including anunsaturated C—C bond between positions 2 and 3 and/or positions 5 and 6,and/or optionally substituted independently positions 2, 3, 4, 5 and 6with a group selected from halo, hydroxy, and methyl;

Ring B is a 6-membered carbocyclic ring, optionally including anunsaturated C—C bond between positions 5 and 10, and/or optionallysubstituted independently at one or more of positions 9 and 10 with agroup selected from halo, hydroxy, and methyl;

Ring C is a 5- or 6-membered carbocyclic ring (i.e., m=0 or 1),optionally substituted at position 10 with a group selected from halo,hydroxy, methyl, ethyl, and carbonyl;

Ring D is a 5-, 6-, or 7-membered carbocyclic ring (i.e., n=0, 1, or 2),optionally including 1, 2, or 3 unsaturated C—C bonds, and/or optionallysubstituted independently as follows:

-   -   at position 14 with a group selected from halo, hydroxy, amino,        carboxy, cyano, nitro, thio, straight-chain or branched        (C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl,        straight-chain or branched (C₁-C₃)alkylamino, and cyclopropyl        bridging to position 12;    -   at position 15 or position 16 with a group selected from halo,        hydroxy, amino, carboxy, cyano, nitro, thio, optionally        substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, optionally        substituted(C₁-C₆)alkyloxy, optionally substituted        (C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,        optionally substituted (C₁-C₆)alkylthio, optionally substituted        (C₁-C₆)alkylsulfonyl, optionally substituted        (C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl,        (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy, optionally        substituted aminocarbonyl, aminocarbonyl(C₁-C₆)alkyl, optionally        substituted cycloalkyl, optionally substituted heterocycloalkyl,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted aryloxy, optionally substituted        arylamino, optionally substituted arylthio, optionally        substituted arylsulfonyl, optionally substituted arylsulfinyl,        optionally substituted aryloxycarbonyl, optionally substituted        arylcarbonyloxy, optionally substituted heteroaryloxy,        optionally substituted heteroarylamino, optionally substituted        heteroarylthio, optionally substituted heteroarylsulfonyl,        optionally substituted heteroarylsulfinyl, optionally        substituted heteroaryloxycarbonyl, optionally substituted        heteroarylcarbonyloxy, alkylaminosulfonyl(C₁-C₆)alkyl,        arylsulfonyl(C₁-C₆)alkyl, and heteroarylsulfonyl(C₁-C₆)alkyl.

Thus, the engineered polypeptides disclosed herein display, amongothers, increased activity, high stereoselectivity, increased solventand thermal stability, and increased product tolerance in the conversionof large prochiral ketone substrate compounds of Formula (II) to thecorresponding chiral amine product compounds of Formula (I).

Accordingly, in one aspect, the present disclosure provides engineeredpolypeptides having transaminase activity, where the engineeredpolypeptide comprises an amino acid sequence having at least 80%identity to SEQ ID NO: 2 and one or more residue differences as comparedto SEQ ID NO:2 at residue positions selected from X19, X21, X34, X53,X56, X73, X86, X88, X107, X113, X147, X155, X165, X171, X178, X233,X251, X259, X268, X277, X286, X312, X316, X317, X358, X366, X383, X399,X414, X415, X417, X426, X434, and X450, wherein the residue differencesat residue positions X21, X56, X86, X88, X107, X113, X133, X147, X233,X286, X312, X316, X383, X415, X417, and X434, are selected from: X21H,X56A/C, X86C, X88H/Y, X107G, X113L/P, X147H/V, X233V, X286C/H, X312N,X316C/F/G/N/S/T, X383C/F/I/M/T, X415A/G/H/L/V, X417V, and X434T. In someembodiments, the residue differences at the residue positions X19, X34,X53, X73, X155, X165, X171, X178, X251, X259, X268, X277, X317, X358,X366, X399, X414, X426, and X450 are selected from X19W, X34A, X53M,X73R, X155V, X165F, X171Q, X178W, X251V, X259V, X268A, X277A, X317L,X358K, X366H, X399A, X414I, X426R, and X450S.

In some embodiments of the engineered polypeptides having transaminaseactivity, the amino acid sequence comprises at least one or more residuedifferences as compared to SEQ ID NO: 2 selected from: X34A, X56A, X88H,X107G, X113L, X147H, X153C, X155V, X233V, X315G, X316N, X383I, andX450S. In some embodiments, the amino acid sequence further comprisesone or more residue differences selected from: X31M, X57F/L, X86N/S,X153A, X233T, X323T, X383V, and X417T.

In some embodiments of the engineered polypeptides having transaminaseactivity, the amino acid sequence comprises at least a combination ofresidue differences as compared to SEQ ID NO: 2 comprising X34A, X56A,X57L, X86S, X88A; X153C, X155V, X163F, X315G, and X417T. In someembodiments, the amino acid sequence further comprises the residuedifference X316N. In some embodiments, the amino acid sequence furthercomprises the residue difference X316N and one or more residuedifferences selected from X31M, X57F, X323T, X383I/T, X415H, and X450S.

In some embodiments of the engineered polypeptides having transaminaseactivity, the amino acid sequence comprises the residue differences ascompared to SEQ ID NO: 2 X34A, X56A, X57L, X86S, X88A; X153C, X155V,X163F, X315G, X316N, and X417T and further comprises a combination ofresidue differences selected from: (a) X31M, X57F, X323T, and X383V; (b)X31M, X57F, X107G, X113L, X233T, X415H, and X450S; (c) X31M, X57F,X233V, X323T, X383I, X415H, and X450S; and (d) X31M, X57F, X147H, X323T,X383I, X415H, and X450S.

In some embodiments of the engineered polypeptides having transaminaseactivity, the engineered polypeptide has at least 1.2 fold increasedstability as compared to the polypeptide of SEQ ID NO: 4, wherein theamino acid sequence comprises one or more residue differences ascompared to SEQ ID NO: 2 selected from: X34T, X107G, X113L, X147H,X155V, X233T/V, X323T, X383I/V, and X450S.

In some embodiments of the engineered polypeptides having transaminaseactivity, the engineered polypeptide has at least 1.2 fold increasedactivity as compared to the polypeptide of SEQ ID NO: 4 in convertingcompound (2) to compound (1), wherein the amino acid sequence comprisesone or more residue differences as compared to SEQ ID NO: 2 selectedfrom: X56A, X86S, X88H, X153C, X415H, and X417T.

In some embodiments of the engineered polypeptides having transaminaseactivity, the engineered polypeptide has increased enantioselectivity ascompared to the polypeptide of SEQ ID NO: 4 in converting compound (2)to compound (1), wherein the amino acid sequence comprises one or moreresidue differences as compared to SEQ ID NO: 2 selected from: X57F,X153C, and X316N.

In some embodiments of the engineered polypeptides having transaminaseactivity, the amino acid sequence further comprises a residue differenceas compared to SEQ ID NO: 2 selected from: X18A, X19W, X21H, X31M, X34A,X53M, X56A/C, X57C/F/L, X73R, X86C/N/S/Y, X88H/Y, X107G, X113C/L/P,X146L, X147H/K/V, X153A/C/V, X155AN, X163L, X165F, X171Q, X178W, X190K,X206K, X228G, X233T/V, X235P, X244T, X251V, X259V, X268A, X277A,X286C/H, X312N, X314N, X315G, X316A/C/F/N/S/T, X317L, X319N, X323T,X358K, X366H, X383C/F/I/L/M/T/V, X395P, X399A, X414I, X415A/G/H/L/V,X417T/V, X424A, X426R, X427Y, X434T, and X450S.

In some embodiments of the engineered polypeptides having transaminaseactivity, the amino acid sequence does not comprise a residue differenceas compared to SEQ ID NO: 2 at positions X9, X45, X177, X211, X294,X324, and X391.

In some embodiments, the engineered transaminase polypeptides can haveadditional residue differences at other residue positions. In someembodiments, the engineered transaminases can have 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25,1-30, 1-35, 1-40, 1-45, or 1-50 additional residue differences ascompared to SEQ ID NO:2. In some embodiments, the engineeredtransaminases can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50additional residue differences. In some embodiments, the amino acidsequence has additionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 18, 20, 21, 22, 23, 24, or 25 residue differences as compared toSEQ ID NO: 2.

Exemplary engineered polypeptides incorporating the residue differences,including various combinations thereof, and having improved properties(e.g., capable of converting compound (2) to compound (1) in at least90% diastereomeric excess under suitable reaction conditions) aredisclosed in Tables 2A and 2B, and the Examples. The amino acidsequences are provided in the Sequence Listing and include SEQ ID NO: 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, and 204.

In another aspect, the present disclosure provides polynucleotidesencoding the engineered polypeptides having transaminase activity, aswell as expression vectors comprising the polynucleotides, and hostcells capable of expressing the polynucleotides encoding the engineeredpolypeptides. Exemplary polynucleotide sequences are provided in theSequence Listing incorporated by reference herein and include SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, and 203.

In some embodiments, the present disclosure also provides methods ofmanufacturing the engineered transaminase polypeptides, where the methodcan comprise culturing a host cell capable of expressing apolynucleotide encoding the engineered transaminase polypeptide underconditions suitable for expression of the polypeptide. In someembodiments, the method for manufacturing the engineered transaminasepolypeptide can also include: (a) synthesizing a polynucleotide encodinga polypeptide comprising an amino acid sequence selected from SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, and 204, and having one or more residuedifferences as compared to SEQ ID NO:2 at residue positions selectedfrom: X19, X21, X34, X53, X56, X73, X86, X88, X107, X113, X147, X155,X165, X171, X178, X233, X251, X259, X268, X277, X286, X312, X316, X317,X358, X366, X383, X399, X414, X415, X417, X426, X434, and X450, whereinthe residue differences at residue positions X21, X56, X86, X88, X107,X113, X133, X147, X233, X286, X312, X316, X383, X415, X417, and X434,are selected from: X21H, X56A/C, X86C, X88H/Y, X107G, X113L/P, X147HN,X233V, X286C/H, X312N, X316C/F/G/N/S/T, X383C/F/I/M/T, X415A/G/H/L/V,X417V, and X434T; and (b) expressing the transaminase polypeptideencoded by the polynucleotide. As noted above, the residue differencesat residue positions X19, X34, X53, X73, X155, X165, X171, X178, X251,X259, X268, X277, X317, X358, X366, X399, X414, X426, and X450 can beselected from X19W, X34A, X53M, X73R, X155V, X165F, X171Q, X178W, X251V,X259V, X268A, X277A, X317L, X358K, X366H, X399A, X414I, X426R, andX450S. As further provided in the detailed description, additionalvariations can be incorporated during the synthesis of thepolynucleotide to prepare engineered transaminases with correspondingdifferences in the expressed amino acid sequences.

The structural features of the engineered transaminase polypeptidesallow for the conversion of large prochiral ketone substrate compounds,other than compound (2), to their corresponding amine product compounds,optionally in stereomeric excess of one chiral amine product overanother chiral amino product. Thus, another aspect of the presentdisclosure are processes using the engineered transaminase polypeptidesto catalyze a reaction in which an amino group from an amino donor istransferred to an amino acceptor, wherein the process comprisescontacting an engineered transaminase polypeptide of the disclosure withan amino acceptor (e.g., a ketone substrate compound) in the presence ofan amino donor (e.g., isopropylamine) under reaction conditions suitablefor converting the amino acceptor to an amine compound.

Accordingly, in some embodiments, the present disclosure provides aprocess for the preparation of an amine compound of Formula (I)

wherein rings A, B, C, and D are as defined above

with the proviso that the compound of Formula (I) is not compound (1)

wherein the method comprises contacting the ketone substrate compound ofFormula (II),

wherein rings A, B, C, and D are as defined above,

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments of the process for preparing an amine compound ofFormula (I), the present disclosure provides a process for preparationof a compound of Formula (Ia)

-   -   wherein

Rings A and B comprise one of the following:

-   -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D comprises an unsaturated C—C bond between positions 12 and 14;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl;

wherein the method comprises contacting the ketone substrate compound ofFormula (IIa),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined abovefor the compound of Formula (Ia),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments of the process for preparing an amine compound ofFormula (I), the present disclosure provides a process for preparationof a compound of Formula (Ib)

wherein

-   -   Rings A and B comprise one of the following:    -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D comprises an unsaturated C—C bond between positions 12 and 14, ora bridging cyclopropyl between positions 12 and 14;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl;

wherein the method comprises contacting the ketone substrate compound ofFormula (IIb),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined abovefor the compound of Formula (Ib),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments of the process for preparing an amine compound ofFormula (I), the present disclosure provides a process for preparationof a compound of Formula (Ic)

wherein

-   -   Rings A and B comprise one of the following:    -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D is aromatic;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl;

wherein the method comprises contacting the ketone substrate compound ofFormula (IIc),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined abovefor the compound of Formula (Ic),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments of the process for preparing an amine compound ofFormula (I), the present disclosure provides a process for preparationof a compound of Formula (Id)

wherein

Ring A comprises an unsaturated C—C bond between positions 2 and 3, orpositions 5 and 6;

R¹ and R² are selected independently from hydrogen, halo, hydroxy,amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionallysubstituted (C₁-C₆)alkylamino, optionally substituted(C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionallysubstituted (C₁-C₆)alkylsulfonyl, optionally substituted(C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl,(C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl,aminocarbonyl(C₁-C₆)alkyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryloxy, optionallysubstituted acylamino, optionally substituted arylthio, optionallysubstituted arylsulfonyl, optionally substituted arylsulfinyl,optionally substituted aryloxycarbonyl, optionally substitutedarylcarbonyloxy, optionally substituted heteroaryloxy, optionallysubstituted heteroarylamino, optionally substituted heteroarylthio,optionally substituted heteroarylsulfonyl, optionally substitutedheteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl,optionally substituted heteroarylcarbonyloxy,alkylaminosulfonyl(C₁-C₆)alkyl, arylsulfonyl(C₁-C₆)alkyl, andheteroarylsulfonyl(C₁-C₆)alkyl;

R³, R⁴, and R⁵ are selected independently from hydrogen, halo, hydroxy,amino, carboxy, cyano, nitro, thio, straight-chain or branched(C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, andstraight-chain or branched (C₁-C₃)alkylamino; and

R⁶, R², and R⁸ are selected independently from hydrogen, halo, hydroxy,and methyl;

wherein the method comprises contacting the ketone substrate compound ofFormula (IId),

wherein R¹, R², R³, R⁴, R⁵, R⁶, R², and R⁸ are as defined above for thecompound of Formula (Id),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments of the processes for preparing the amine compoundsof the present disclosure, the stereoselectivity of the transaminasesprovides for the preparation of the chiral amine compounds of Formula(I), Formula (Ia), Formula (Ib), Formula (Ic), and Formula (Id) indiastereomeric excess. In some embodiments, the process results in theformation of the chiral amine compound of Formula (I), Formula (Ia),Formula (Ib), Formula (Ic), and Formula (Id) in diastereomeric excess ofat least 90%, 95%, 96%, 97%, 98%, 99%, or greater.

As provided herein, the processes using the engineered transaminases canbe done under a range of suitable reaction conditions, including, amongothers, ranges of amine donor, pH, temperature, buffer, solvent system,substrate loading, polypeptide loading, cofactor loading, pressure, andreaction time.

In some embodiments, the suitable reaction conditions for thetransamination process can comprise: (a) substrate loading at about 5g/L to 200 g/L; (b) about 0.1 to 50 g/L of engineered transaminasepolypeptide; (c) about 0.1 to 4 M of isopropylamine (IPM); (d) about 0.1to 10 g/L of pyridoxal phosphate (PLP) cofactor; (e) pH of about 6 to 9;and (f) temperature of about 30 to 60° C.

In some embodiments, the suitable reaction conditions for thetransamination process can comprise: (a) substrate loading at about 10g/L to 150 g/L; (b) about 0.5 to 20 g/L of engineered transaminasepolypeptide; (c) about 0.1 to 3 M of isopropylamine (IPM); (d) about 0.1to 10 g/L of pyridoxal phosphate (PLP) cofactor; (e) about 0.05 to 0.20M TEA buffer; (f) about 1% to about 45% DMSO; (g) pH of about 6 to 9;and (h) temperature of about 30 to 65° C.

In some embodiments, the suitable reaction conditions for thetransamination process can comprise: (a) substrate loading at about 20to 100 g/L; (b) about 1 to 5 g/L of engineered transaminase polypeptide;(c) about 0.5 to 2 M of isopropylamine (IPM); (d) about 0.2 to 2 g/L ofpyridoxal phosphate (PLP) cofactor; (e) about 0.1 M TEA buffer; (f)about 25% DMSO; (e) pH of about 8; and (f) temperature of about 45 to60° C.

Guidance on the choice of engineered transaminases, preparation of thebiocatalysts, the choice of enzyme substrates, and parameters forcarrying out the processes are further described in the detaileddescription that follow.

5. DETAILED DESCRIPTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “apolypeptide” includes more than one polypeptide.

Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

It is to be understood that both the foregoing general description,including the drawings, and the following detailed description areexemplary and explanatory only and are not restrictive of thisdisclosure.

The section headings used herein are for organizational purposes onlyand not to be construed as limiting the subject matter described.

5.1 Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

5.2 Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotide” or “nucleic acid’ refers to two or more nucleosidesthat are covalently linked together. The polynucleotide may be whollycomprised ribonucleosides (i.e., an RNA), wholly comprised of2′-deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and2′-deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), itmay include one or more modified and/or synthetic nucleobases, such as,for example, inosine, xanthine, hypoxanthine, etc. Preferably, suchmodified or synthetic nucleobases will be encoding nucleobases.

“Aminotransferase” and “transaminase” are used interchangeably herein torefer to a polypeptide having an enzymatic capability of transferring anamino group (NH₂) from a primary amine to a carbonyl group (C═O) of anacceptor molecule. Transaminases as used herein include naturallyoccurring (wild-type) transaminases as well as non-naturally occurringengineered polypeptides generated by human manipulation.

“Amino acceptor” and “amine acceptor,” “keto substrate,” “keto,” and“ketone” are used interchangeably herein to refer to a carbonyl (keto,or ketone) compound which accepts an amino group from a donor amine. Insome embodiments, amino acceptors are molecules of the following generalformula,

in which each of R^(α) and R^(β), when taken independently, is an alkyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, which can beunsubstituted or substituted with one or more enzymatically acceptablegroups. R^(α) may be the same or different from R^(β) in structure orchirality. In some embodiments, R^(α) and R^(β), taken together, mayform a ring that is unsubstituted, substituted, or fused to other rings.Amino acceptors include keto carboxylic acids and alkanones (ketones).Typical keto carboxylic acids are α-keto carboxylic acids such asglyoxalic acid, pyruvic acid, oxaloacetic acid, and the like, as well assalts of these acids Amino acceptors also include substances which areconverted to an amino acceptor by other enzymes or whole cell processes,such as fumaric acid (which can be converted to oxaloacetic acid),glucose (which can be converted to pyruvate), lactate, maleic acid, andothers. Amino acceptors that can be used include, by way of example andnot limitation, 3,4-dihydronaphthalen-1(2H)-one, 1-phenylbutan-2-one,3,3-dimethylbutan-2-one, octan-2-one, ethyl 3-oxobutanoate,4-phenylbutan-2-one, 1-(4-bromophenyflethanone, 2-methyl-cyclohexamone,7-methoxy-2-tetralone, 1-hydroxybutan-2-one, pyruvic acid, acetophenone,3′-hydroxyacetophenone, 2-methoxy-5-fluoroacetophenone, levulinic acid,1-phenylpropan-1-one, 1-(4-bromophenyl)propan-1-one,1-(4-nitrophenyl)propan-1-one, 1-phenylpropan-2-one,2-oxo-3-methylbutanoic acid,1-(3-trifluoromethylphenyl)propan-1-one,hydroxypropanone,methoxyoxypropanone, 1-phenylbutan-1-one,1-(2,5-dimethoxy-4-methylphenyl)butan-2-one,1-(4-hydroxyphenyl)butan-3-one, 2-acetylnaphthalene, phenylpyruvic acid,2-ketoglutaric acid, and 2-ketosuccinic acid, including both (R) and (S)single isomers where possible.

“Amino donor” or “amine donor” refers to an amino compound which donatesan amino group to the amino acceptor, thereby becoming a carbonylspecies. In some embodiments, amino donors are molecules of thefollowing general formula,

in which each of R^(ε) and R^(δ), when taken independently, is an alkyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, which isunsubstituted or substituted with one or more enzymaticallynon-inhibiting groups. R^(ε) can be the same or different from R^(δ) instructure or chirality. In some embodiments, R^(ε) and R^(δ), takentogether, may form a ring that is unsubstituted, substituted, or fusedto other rings. Typical amino donors that can be used include chiral andachiral amino acids, and chiral and achiral amines. Amino donors thatcan be used include, by way of example and not limitation,isopropylamine (also referred to as 2-aminopropane), α-phenethylamine(also termed 1-phenylethanamine), and its enantiomers(S)-1-phenylethanamine and (R)-1-phenylethanamine,2-amino-4-phenylbutane, glycine, L-glutamic acid, L-glutamate,monosodium glutamate, L-alanine, D-alanine, D,L-alanine, L-asparticacid, L-lysine, D,L-ornithine, β-alanine, taurine, n-octylamine,cyclohexylamine, 1,4-butanediamine (also referred to as putrescine),1,6-hexanediamine, 6-aminohexanoic acid, 4-aminobutyric acid, tyramine,and benzyl amine, 2-aminobutane, 2-amino-1-butanol,1-amino-1-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane,1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane,1-amino-1-(4-bromophenyl)propane, 1-amino-1-(4-nitrophenyl)propane,1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane,2-aminopropanol, 1-amino-1-phenylbutane, 1-phenyl-2-aminobutane,1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methylcyclopentane,1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane,1-amino-1-(2-naphthyl)ethane, 3-methylcyclopentylamine,2-methylcyclopentylamine, 2-ethylcyclopentylamine,2-methylcyclohexylamine, 3-methylcyclohexylamine, 1-aminotetralin,2-aminotetralin, 2-amino-5-methoxytetralin, and 1-aminoindan, includingboth (R) and (S) single isomers where possible and including allpossible salts of the amines.

“Chiral amine” refers to amines of general formula R^(α)—CH(NH₂)—R^(β)and is employed herein in its broadest sense, including a wide varietyof aliphatic and alicyclic compounds of different, and mixed, functionaltypes, characterized by the presence of a primary amino group bound to asecondary carbon atom which, in addition to a hydrogen atom, carrieseither (i) a divalent group forming a chiral cyclic structure, or (ii)two substituents (other than hydrogen) differing from each other instructure or chirality. Divalent groups forming a chiral cyclicstructure include, for example, 2-methylbutane-1,4-diyl,pentane-1,4-diyl,hexane-1,4-diyl, hexane-1,5-diyl,2-methylpentane-1,5-diyl. The two different substituents on thesecondary carbon atom (R^(α) and R^(β) above) also can vary widely andinclude alkyl, aralkyl, aryl, halo, hydroxy, lower alkyl, lower alkoxy,lower alkylthio, cycloalkyl, carboxy, carbalkoxy, carbamoyl, mono- anddi-(lower alkyl) substituted carbamoyl, trifluoromethyl, phenyl, nitro,amino, mono- and di-(lower alkyl) substituted amino, alkylsulfonyl,arylsulfonyl, alkylcarboxamido, arylcarboxamido, etc., as well as alkyl,aralkyl, or aryl substituted by the foregoing.

“Pyridoxal-phosphate,” “PLP,” “pyridoxal-5′-phosphate,” “PYP,” and “P5P”are used interchangeably herein to refer to the compound that acts as acoenzyme in transaminase reactions. In some embodiments, pyridoxalphosphate is defined by the structure1-(4′-formyl-3′-hydroxy-2′-methyl-5′-pyridyl)methoxyphosphonic acid, CASnumber [54-47-7]. Pyridoxal-5′-phosphate can be produced in vivo byphosphorylation and oxidation of pyridoxol (also known as Vitamin B₆).In transamination reactions using transaminase enzymes, the amine groupof the amino donor is transferred to the coenzyme to produce a ketobyproduct, while pyridoxal-5′-phosphate is converted to pyridoxaminephosphate. Pyridoxal-5′-phosphate is regenerated by reaction with adifferent keto compound (the amino acceptor). The transfer of the aminegroup from pyridoxamine phosphate to the amino acceptor produces anamine and regenerates the coenzyme. In some embodiments, thepyridoxal-5′-phosphate can be replaced by other members of the vitaminB₆ family, including pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM),and their phosphorylated counterparts; pyridoxine phosphate (PNP), andpyridoxamine phosphate (PMP).

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)). Examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence. For instance, a “reference sequence based on SEQID NO:2 having at the residue corresponding to X34 an alanine” or X34Arefers to a reference sequence in which the corresponding residue at X34in SEQ ID NO:2, which is a threonine, has been changed to alanine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequencefor optimal alignment of the two sequences. The comparison window can belonger than 20 contiguous residues, and includes, optionally 30, 40, 50,100, or longer windows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredtransaminase, can be aligned to a reference sequence by introducing gapsto optimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a change inthe amino acid residue at a position of a polypeptide sequence relativeto the amino acid residue at a corresponding position in a referencesequence. The positions of amino acid differences generally are referredto herein as “Xn,” where n refers to the corresponding position in thereference sequence upon which the residue difference is based. Forexample, a “residue difference at position X34 as compared to SEQ ID NO:2” refers to a change of the amino acid residue at the polypeptideposition corresponding to position 34 of SEQ ID NO:2. Thus, if thereference polypeptide of SEQ ID NO: 2 has a threonine at position 34,then a “residue difference at position X34 as compared to SEQ ID NO:2”an amino acid substitution of any residue other than threonine at theposition of the polypeptide corresponding to position 34 of SEQ ID NO:2. In most instances herein, the specific amino acid residue differenceat a position is indicated as “XnY” where “Xn” specified thecorresponding position as described above, and “Y” is the single letteridentifier of the amino acid found in the engineered polypeptide (i.e.,the different residue than in the reference polypeptide). In someembodiments, where more than one amino acid can appear in a specifiedresidue position, the alternative amino acids can be listed in the formXnY/Z, where Y and Z represent alternate amino acid residues. In someinstances (e.g., in Tables 2A and 2B), the present disclosure alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide.Furthermore, in some instances, a polypeptide of the present disclosurecan include one or more amino acid residue differences relative to areference sequence, which is indicated by a list of the specifiedpositions where changes are made relative to the reference sequence.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid, e.g., alanine, valine, leucine, and isoleucine; an amino acid withhydroxyl side chain is substituted with another amino acid with ahydroxyl side chain, e.g., serine and threonine; an amino acid havingaromatic side chains is substituted with another amino acid having anaromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, andhistidine; an amino acid with a basic side chain is substituted withanother amino acid with a basic side chain, e.g., lysine and arginine;an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain, e.g., aspartic acid or glutamicacid; and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively. Exemplaryconservative substitutions are provided in Table 1 below.

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine), (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered transaminase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered transaminase enzymes comprise insertions of oneor more amino acids to the naturally occurring transaminase polypeptideas well as insertions of one or more amino acids to other improvedtransaminase polypeptides. Insertions can be in the internal portions ofthe polypeptide, or to the carboxy or amino terminus. Insertions as usedherein include fusion proteins as is known in the art. The insertion canbe a contiguous segment of amino acids or separated by one or more ofthe amino acids in the reference polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length transaminase polypeptide,for example the reference engineered transaminase polypeptide of SEQ IDNO: 2.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved transaminase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved transaminase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis, it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure transaminase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved transaminase polypeptide is a substantially pure polypeptidecomposition.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate, e.g., compound (2), to itscorresponding chiral amine product, e.g., compound (1), with at leastabout 85% stereomeric excess.

“Improved enzyme property” refers to a transaminase polypeptide thatexhibits an improvement in any enzyme property as compared to areference transaminase. For the engineered transaminase polypeptidesdescribed herein, the comparison is generally made to the wild-typetransaminase enzyme, although in some embodiments, the referencetransaminase can be another engineered transaminase. Enzyme propertiesfor which improvement is desirable include, but are not limited to,enzymatic activity (which can be expressed in terms of percentconversion of the substrate), thermo stability, solvent stability, pHactivity profile, cofactor requirements, refractoriness to inhibitors(e.g., substrate or product inhibition), and stereoselectivity(including enantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered transaminase polypeptides, which can be represented by anincreased specific activity (e.g., product produced/time/weight protein)or an increased percent conversion of the substrate to the product(e.g., percent conversion of starting amount of substrate to product ina specified time period using a specified amount of transaminase) ascompared to the reference transaminase enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max), or k_(cal), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.2 fold the enzymatic activity of the correspondingwild-type transaminase enzyme, to as much as 2 fold, 5 fold, 10 fold, 20fold, 25 fold, 50 fold, 75 fold, 100 fold, or more enzymatic activitythan the naturally occurring transaminase or another engineeredtransaminase from which the transaminase polypeptides were derived.Transaminase activity can be measured by any one of standard assays,such as by monitoring changes in spectrophotometric properties ofreactants or products. In some embodiments, the amount of productsproduced can be measured by High-Performance Liquid Chromatography(HPLC) separation combined with UV absorbance or fluorescent detectionfollowing derivatization, such as with o-phthaldialdehyde (OPA).Comparisons of enzyme activities are made using a defined preparation ofenzyme, a defined assay under a set condition, and one or more definedsubstrates, as further described in detail herein. Generally, whenlysates are compared, the numbers of cells and the amount of proteinassayed are determined as well as use of identical expression systemsand identical host cells to minimize variations in amount of enzymeproduced by the host cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate(s) tothe corresponding product(s). “Percent conversion” refers to the percentof the substrate that is converted to the product within a period oftime under specified conditions. Thus, the “enzymatic activity” or“activity” of a transaminase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” refers to a transaminase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the wild-type enzyme.

“Solvent stable” refers to a transaminase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to varyingconcentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol,dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran,acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for aperiod of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme.

“Thermo- and solvent stable” refers to a transaminase polypeptide thatis both thermostable and solvent stable.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporated herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered transaminase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5× SSPE, 0.2% SDS at 42° C., followed by washing in 0.2× SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5× SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1× SSC containing 0.1% SDS at 65° C.Other high stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding thetransaminase enzymes may be codon optimized for optimal production fromthe host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refer to those conditions in thebiocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which a transaminase polypeptide of the presentdisclosure is capable of converting a substrate compound to a productcompound (e.g., conversion of compound (2) to compound (1)). Exemplary“suitable reaction conditions” are provided in the detailed descriptionand illustrated by the Examples.

“Loading”, such as in “compound loading” or “enzyme loading” or“cofactor loading” refers to the concentration or amount of a componentin a reaction mixture at the start of the reaction.

“Substrate” in the context of a biocatalyst mediated process refers tothe compound or molecule acted on by the biocatalyst. For example, anexemplary substrate for the engineered transaminase biocatalysts in theprocess disclosed herein is compound (2).

“Product” in the context of a biocatalyst mediated process refers to thecompound or molecule resulting from the action of the biocatalyst. Forexample, an exemplary product for the engineered transaminasebiocatalysts in the process disclosed herein is compound (1).

“Heteroalkyl, “heteroalkenyl,” and “heteroalkynyl,” refer to alkyl,alkenyl and alkynyl as defined herein in which one or more of the carbonatoms are each independently replaced with the same or differentheteroatoms or heteroatomic groups. Heteroatoms and/or heteroatomicgroups which can replace the carbon atoms include, but are not limitedto, —O—, —S—, —S—O—, —NR^(γ)—, —PH—, —S(O)—, —S(O)2-, —S(O)NR^(γ)—,—S(O)₂NR^(γ)—, and the like, including combinations thereof, where eachR^(γ) is independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and other suitablesubstituents.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to12 carbon atoms inclusively having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Exemplary arylsinclude phenyl, pyridyl, naphthyl and the like.

“Arylalkyl” refers to an alkyl substituted with an aryl, i.e.,aryl-alkyl-groups, preferably having from 1 to 6 carbon atomsinclusively in the alkyl moiety and from 6 to 12 carbon atomsinclusively in the aryl moiety. Such arylalkyl groups are exemplified bybenzyl, phenethyl and the like.

“Arylalkenyl” refers to an alkenyl substituted with an aryl, i.e.,aryl-alkenyl-groups, preferably having from 2 to 6 carbon atomsinclusively in the alkenyl moiety and from 6 to 12 carbon atomsinclusively in the aryl moiety.

“Arylalkynyl” refers to an alkynyl substituted with an aryl, i.e.,aryl-alkynyl-groups, preferably having from 2 to 6 carbon atomsinclusively in the alkynyl moiety and from 6 to 12 carbon atomsinclusively in the aryl moiety.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 12 carbon atomsinclusively having a single cyclic ring or multiple condensed ringswhich can be optionally substituted with from 1 to 3 alkyl groups.Exemplary cycloalkyl groups include, but are not limited to, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and thelike, or multiple ring structures, including bridged ring systems, suchas adamantyl, and the like.

“Cycloalkylalkyl” refers to an alkyl substituted with a cycloalkyl,i.e., cycloalkyl-alkyl-groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 3 to 12 carbon atomsinclusively in the cycloalkyl moiety. Such cycloalkylalkyl groups areexemplified by cyclopropylmethyl, cyclohexylethyl and the like.

“Cycloalkylalkenyl” refers to an alkenyl substituted with a cycloalkyl,i.e., cycloalkyl-alkenyl-groups, preferably having from 2 to 6 carbonatoms inclusively in the alkenyl moiety and from 3 to 12 carbon atomsinclusively in the cycloalkyl moiety.

“Cycloalkylalkynyl” refers to an alkynyl substituted with a cycloalkyl,i.e., cycloalkyl-alkynyl-groups, preferably having from 2 to 6 carbonatoms inclusively in the alkynyl moiety and from 3 to 12 carbon atomsinclusively in the cycloalkyl moiety.

“Amino” refers to the group —NH₂. Substituted amino refers to the group—NHR^(η), NR^(η)R^(η), and NR^(η)R^(η)R^(η), where each R^(η) isindependently selected from substituted or unsubstituted alkyl,cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl,acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like.Typical amino groups include, but are limited to, dimethylamino,diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino,furanyl-oxy-sulfamino, and the like.

“Alkylamino” refers to a —NHR^(ζ) group, where R^(ζ) is an alkyl, anN-oxide derivative, or a protected derivative thereof, e.g.,methylamino, ethylamino, n-propylamino, iso-propylamino, n-butylamino,iso-butylamino, tert-butylamino, or methylamino-N-oxide, and the like.

“Arylamino” refers to —NHR^(λ), where R^(λ) is an aryl group, which canbe optionally substituted.

“Heteroarylamino” refers to —NHR^(σ) where R^(σ) is a heteroaryl group,which can be optionally substituted.

“Aminoalkyl” refers to an alkyl group in which one or more of thehydrogen atoms is replaced with an amino group, including a substitutedamino group.

“Oxo” refers to ═O

“Oxy” refers to a divalent group —O—, which may have varioussubstituents to form different oxy groups, including ethers and esters.

“Alkoxy” or “alkyloxy” are used interchangeably herein to refer to thegroup —OR^(ζ), wherein R^(ζ) is an alkyl group, including optionallysubstituted alkyl groups as also defined herein.

“Aryloxy” refers to —OR^(λ) groups, where R^(λ) is an aryl group, whichcan be optionally substituted.

“Heteroaryloxy” refers to —OR^(σ), where R^(σ) is a heteroaryl group,which can be optionally substituted.

“Carboxy” refers to —COOH.

“Carboxyalkyl” refers to an alkyl substituted with a carboxy group.

“Carbonyl” refers to —C(O)—, which may have a variety of substituents toform different carbonyl groups including acids, acid halides, aldehydes,amides, esters, and ketones.

“Alkylcarbonyl” refers to —C(O)R^(ζ), where R^(ζ) is an alkyl group,which can be optionally substituted.

“Arylcarbonyl” refers to —C(O)R^(λ), where R^(λ) is an aryl group, whichcan be optionally substituted.

“Heteroarylcarbonyl” refers to —C(O)R^(σ) where R^(σ) is a heteroarylgroup, which can be optionally substituted.

“Alkyloxycarbonyl” refers to —C(O)OR^(ζ), where R^(ζ) is an alkyl group,which can be optionally substituted.

“Aryloxycarbonyl” refers to —C(O)OR^(λ), where R^(λ) is an aryl group,which can be optionally substituted.

“Heteroaryloxycarbonyl” refers to —C(O)OR^(σ), where R^(σ) is aheteroaryl group, which can be optionally substituted.

“Arylalkyloxycarbonyl” refers to —C(O)OR^(ρ), where R^(ρ) is anaryl-alkyl-group, which can be optionally substituted.

“Alkylcarbonyloxy” refers to —OC(O)—R^(ζ), where R is an alkyl group,which can be optionally substituted.

“Arylcarbonyloxy” refers to —OC(O)R^(λ), where R is an aryl group, whichcan be optionally substituted.

“Heteroarylalkyloxycarbonyl” refers to —C(O)OR^(ω), where R^(ω) is aheteroarylalkyl group, which can be optionally substituted.

“Heteroarylcarbonyloxy” refers to —OC(O)R^(σ), where R^(σ) is anheteroaryl group, which can be optionally substituted.

“Aminocarbonyl” refers to —C(O)NH₂. Substituted aminocarbonyl refers to—C(O)NR^(η)R^(η), where the amino group NR^(η)R^(η) is as definedherein.

“Aminocarbonylalkyl” refers to an alkyl substituted with anaminocarbonyl group.

“Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

“Haloalkyl” refers to an alkyl group substituted with one or morehalogen. Thus, the term “haloalkyl” is meant to include monohaloalkyls,dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, theexpression “(C₁ C₂) haloalkyl” includes 1-fluoromethyl, difluoromethyl,trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl,1,1,1 trifluoroethyl, perfluoroethyl, etc.

“Hydroxy” refers to —OH.

“Hydroxyalkyl” refers to an alkyl substituted with one or more hydroxygroup.

“Cyano” refers to —CN.

“Nitro” refers to —NO₂.

“Thio” or “sulfanyl” refers to —SH. Substituted thio or sulfanyl refersto —S—R^(η), where R^(η) is an alkyl, aryl or other suitablesubstituent.

“Alkylthio” refers to —SR^(ζ), where R^(ζ) is an alkyl, which can beoptionally substituted. Typical alkylthio group include, but are notlimited to, methylthio, ethylthio, n-propylthio, and the like.

“Arylthio” refers to —SR^(λ), where R^(λ) is an aryl, which can beoptionally substituted. Typical arylthio groups include, but are notlimited to, phenylthio, (4-methylphenyl)thio, pyridinylthio, and thelike.

“Heteroarylthio” refers to —SR^(σ), where R^(σ) is a heteroaryl, whichcan be optionally substituted.

“Sulfonyl” refers to —SO₂—. Substituted sulfonyl refers to —SO₂—R^(η),where R^(η) is an alkyl, aryl or other suitable substituent.

“Alkylsulfonyl” refers to —SO₂—R^(ζ), where R^(ζ) is an alkyl, which canbe optionally substituted. Typical alkylsulfonyl groups include, but arenot limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and thelike.

“Arysulfonyl” refers to —SO₂—R^(λ), where R^(λ) is an aryl, which can beoptionally substituted. Typical arylsulfonyl groups include, but are notlimited to, phenylsulfonyl, (4-methylphenyl)sulfonyl, pyridinylsulfonyl,and the like.

“Heteroarylsulfonyl” refers to —SO₂—R^(σ), where R^(σ) is a heteroarylgroup, which can be optionally substituted.

“Sulfinyl” refers to —SO—. Substituted sulfinyl refers to —SO—R^(η),where R^(η) is an alkyl, aryl or other suitable substituent.

“Alkylsulfinyl” refers to —SO—R^(ζ), where R^(ζ) is an alkyl, which canbe optionally substituted. Typical alkylsulfinyl groups include, but arenot limited to, methylsulfinyl, ethylsulfinyl, n-propylsulfinyl, and thelike.

“Arysulfinyl” refers to —SO—R^(λ), where R^(λ) is an aryl, which can beoptionally substituted. Typical arylsulfinyl groups include, but are notlimited to, phenylsulfinyl, (4-methylphenyl)sulfinyl, pyridinylsulfinyl,and the like.

“Heteroarylsulfinyl” refers to —SO—R^(σ), where R^(σ) is a heteroarylgroup, which can be optionally substituted.

“Alkylaminosulfonylalkyl” refers to an alkyl substituted with analkyl-NH—SO₂— group.

“Arylsulfonylalkyl” refers to an alkyl substituted with an aryl-SO₂—group.

“Heteroarylsulfonylalkyl” refers to an alkyl substituted with aheteroaryl-SO₂— group.

“Aminosulfonyl” refers to —SO₂NH₂. Substituted aminosulfonyl refers to—SO₂NR^(δ)R^(δ), where the amino group —NR^(η)R^(η) is as definedherein.

“Heteroaryl” refers to an aromatic heterocyclic group of from 1 to 10carbon atoms inclusively and 1 to 4 heteroatoms inclusively selectedfrom oxygen, nitrogen and sulfur within the ring. Such heteroaryl groupscan have a single ring (e.g., pyridyl or furyl) or multiple condensedrings (e.g., indolizinyl or benzothienyl).

“Heteroarylalkyl” refers to an alkyl substituted with a heteroaryl,i.e., heteroaryl-alkyl-groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 5 to 12 ring atomsinclusively in the heteroaryl moiety. Such heteroarylalkyl groups areexemplified by pyridylmethyl and the like.

“Heteroarylalkenyl” refers to an alkenyl substituted with a heteroaryl,i.e., heteroaryl-alkenyl-groups, preferably having from 2 to 6 carbonatoms inclusively in the alkenyl moiety and from 5 to 12 ring atomsinclusively in the heteroaryl moiety.

“Heteroarylalkynyl” refers to an alkynyl substituted with a heteroaryl,i.e., heteroaryl-alkynyl-groups, preferably having from 2 to 6 carbonatoms inclusively in the alkynyl moiety and from 5 to 12 ring atomsinclusively in the heteroaryl moiety.

“Heterocycle”, “heterocyclic” and interchangeably “heterocycloalkyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, from 2 to 10 carbon ring atoms inclusively andfrom 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfuror oxygen within the ring. Such heterocyclic groups can have a singlering (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings(e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples ofheterocycles include, but are not limited to, furan, thiophene,thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, pyrrolidine, indoline and the like.

“Heterocycloalkylalkyl” refers to an alkyl substituted with aheterocycloalkyl, i.e., heterocycloalkyl-alkyl-groups, preferably havingfrom 1 to 6 carbon atoms inclusively in the alkyl moiety and from 3 to12 ring atoms inclusively in the heterocycloalkyl moiety.

“Heterocycloalkylalkenyl” refers to an alkenyl substituted with aheterocycloalkyl, i.e., heterocycloalkyl-alkenyl-groups, preferablyhaving from 2 to 6 carbon atoms inclusively in the alkenyl moiety andfrom 3 to 12 ring atoms inclusively in the heterocycloalkyl moiety.

“Heterocycloalkylalkynyl” refers to an alkynyl substituted with aheterocycloalkyl, i.e., heterocycloalkyl-alkynyl-groups, preferablyhaving from 2 to 6 carbon atoms inclusively in the alkynyl moiety andfrom 3 to 12 ring atoms inclusively in the heterocycloalkyl moiety.

“Leaving group” generally refers to any atom or moiety that is capableof being displaced by another atom or moiety in a chemical reaction.More specifically, a leaving group refers to an atom or moiety that isreadily displaced and substituted by a nucleophile (e.g., an amine, athiol, an alcohol, or cyanide). Such leaving groups are well known andinclude carboxylates, N-hydroxysuccinimide (“NHS”),N-hydroxybenzotriazole, a halogen (fluorine, chlorine, bromine, oriodine), and alkyloxy groups. Non-limiting characteristics and examplesof leaving groups can be found, for example in Organic Chemistry, 2ded., Francis Carey (1992), pages 328-331; Introduction to OrganicChemistry, 2d ed., Andrew Streitwieser and Clayton Heathcock (1981),pages 169-171; and Organic Chemistry, 5th Ed., John McMurry, Brooks/ColePublishing (2000), pages 398 and 408; all of which are incorporatedherein by reference.

Unless otherwise specified, positions occupied by hydrogen in theforegoing groups can be further substituted with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy,alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present disclosure, and is otherwise chemically reasonable.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. One of ordinary skill in the art would understand that withrespect to any molecule described as containing one or more optionalsubstituents, only sterically practical and/or synthetically feasiblecompounds are meant to be included. “Optionally substituted” refers toall subsequent modifiers in a term or series of chemical groups. Forexample, in the term “optionally substituted arylalkyl, the “alkyl”portion and the “aryl” portion of the molecule may or may not besubstituted, and for the series “optionally substituted alkyl,cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, andheteroaryl groups, independently of the others, may or may not besubstituted.

“Protecting group” refers to a group of atoms that mask, reduce orprevent the reactivity of the functional group when attached to areactive functional group in a molecule. Typically, a protecting groupmay be selectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Wuts and Greene, “Greene'sProtective Groups in Organic Synthesis,” 4^(th) Ed., Wiley Interscience(2006), and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Functional groups that canhave a protecting group include, but are not limited to, hydroxy, amino,and carboxy groups. Representative amino protecting groups include, butare not limited to, formyl, acetyl, trifluoroacetyl, benzyl,benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl(“TMS”), 2-trimethylsilyl-ethanesulfonyl (“SES”), trityl and substitutedtrityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”),nitro-veratryloxycarbonyl (“NVOC”) and the like.

“Polyol” as used herein refers to compounds containing multiple hydroxygroups. In reference to polymers, polyol includes polymers with hydroxylfunctional groups. Exemplary polymeric polyols include, by way ofexample and not limitation, polyethers and polyesters, e.g.,polyethylene glycol, polypropylene glycol, poly(tetramethylene) glycoland polytetrahydrofuran.

5.3 Engineered Transaminase Polypeptides

The present disclosure provides engineered polypeptides havingtransaminase activity, polynucleotides encoding the polypeptides, andmethods for using the polypeptides. Where the foregoing descriptionrelates to polypeptides, it is to be understood that it also describesthe polynucleotides encoding the polypeptides.

Transaminases, also known as aminotransferases, catalyze the transfer ofan amino group from a primary amine of an amino donor substrate to thecarbonyl group (e.g., a keto or aldehyde group) of an amino acceptormolecule. Transaminases have been identified from a variety ofmicroorganisms including but not limited to Alcaligenes denitrificans,Bordetella bronchiseptica, Bordetella parapertussis, Brucellamelitensis, Burkholderia malle, Burkholderia pseudomallei,Chromobacterium violaceum, Oceanicola granulosus HTCC2516, Oceanobactersp. RED65, Oceanospirillum sp. MED92, Pseudomonas putida, Ralstoniasolanacearum, Rhizobium meliloti, Rhizobium sp. (strain NGR234),Bacillus thuringensis, Klebsiella pneumoniae and Vibrio fluvialis (seee.g., Shin et al., 2001, Biosci. Biotechnol, Biochem. 65:1782-1788).

Transaminases are useful for the chiral resolution of racemic amines byexploiting the ability of the transaminases to carry out the reaction ina stereospecific manner, i.e., preferential conversion of one enantiomerto the corresponding ketone, thereby resulting in a mixture enriched inthe other enantiomer (see, e.g., Koselewski et al., 2009, Org Lett.11(21):4810-2). The stereoselectivity of transaminases in the conversionof a ketone to the corresponding amine also make these enzymes useful inthe asymmetric synthesis of optically pure amines from the correspondingketo compounds (see, e.g., Höhne et al., “Biocatalytic Routes toOptically Active Amines,” Chem Cat Chem 1(1):42-51; Zua and Hua, 2009,Biotechnol J. 4(10):1420-31).

The wild-type ω-transaminase from Vibrio fluvialis ω-VfT displays highenantioselectivity for (S)-enantiomers of certain chiral amines and hassubstrate specificity for chiral aromatic amines (see e.g., Shin andKim, 2002, J. Org. Chem. 67:2848-2853). The high enantioselectivity ofω-VfT has been applied to chiral resolution of amines (see e.g., Yun, etal., 2004, Biotechnol. Bioeng. 87:772-778; Shin and Kim, 1997,Biotechnol. Bioeng. 55:348-358; M. Hçhne, et al., 2008, Adv. Synth.Catal. 350:802-807). The ω-VfT transaminase has also been used in theasymmetric synthesis of optically pure amines using a prochiral ketonesubstrate. However, the use of this transaminase in asymmetric synthesisof chiral amines is limited by the unfavorable equilibrium of thereverse reaction (see e.g., Shin and Kim, 1999, Biotechnol. Bioeng. 65,206-211); inhibition of by the chiral amine product (see e.g., Shin etal., 2001, Biotechnol Bioeng 73:179-187; Yun and Kim, 2008, Biosci.Biotechnol. Biochem. 72(11):3030-3033); low activity on amine acceptorshaving bulky side chains, such as aromatic groups (see e.g., Shin andKim, 2002, J. Org. Chem. 67:2848-2853); and low enzyme stability (seee.g., Yun and Kim, supra).

Variant transaminases derived from the ω-VfT transaminase of Vibriofluvialis have been reported that have increased resistance to aliphaticketones (see e.g., Yun et al., 2005, Appl Environ Micriobiol.71(8):4220-4224) and broadened amino donor substrate specificity (seee.g., Cho et al., 2008, Biotechnol Bioeng. 99(2):275-84). Patentpublications WO2010081053 and US20100209981 (each of which is herebyincorporated by reference herein) describe engineered transaminasesderived from ω-VfT that have improved properties for use in synthesis ofchiral amine compounds including increased stability to temperatureand/or organic solvent, and increased enzymatic activity towardsstructurally different amino acceptor molecules. Patent publicationWO2011159910 (which is hereby incorporated by reference herein)describes engineered transaminases derived from ω-VfT that are optimizedfor the enantioselective conversion of the substrate3′-hydroxyacetophenone to the product (S)-3-(1-aminoethyl)-phenol.

The present disclosure relates to engineered transaminase polypeptidesderived from the previously engineered transaminases disclosed in patentpublication WO2010081053. The engineered transaminases of the presentdisclosure have been engineered with amino acid residue substitutionsthat allow for conversion of particularly large amino acceptor compoundsubstrates to the corresponding chiral amine compound products.

Significantly, the present disclosure identifies amino acid residuepositions and corresponding amino acid residue substitutions in theengineered transaminase polypeptide that can increase the enzymaticactivity, enantioselectivity, stability, and refractoriness to productinhibition, with these particularly large amine acceptor substrates.

The identification of the specific residue positions and substitutionsin the engineered transaminase polypeptides of the present disclosure byengineering through directed evolution methods using structure-basedrational sequence library design with screening for improved functionalproperties using an activity assay based on the conversion of theprochiral ketone group of an exemplary large substrate amine acceptor ofcompound to its corresponding chiral amine product. Specifically, theconversion of the ketone of the cyclopamine analog compound of compound(2) to the corresponding chiral amine compound of compound (1), as shownin Scheme 3.

The engineered transaminase polypeptides of the present disclosure wereevolved to efficiently convert the ketone of the exemplary substratecompound (2) to the corresponding chiral amine of the exemplary productcompound (1), in the presence of an amino donor under suitable reactionconditions, and in diasteriomeric excess (i.e., in excess of otherdiastereomers having the opposite enantiomer at the chiral aminecenter).

The specific structural features and structure-function correlatinginformation of the engineered transaminase polypeptides of the presentdisclosure also allows for engineered transaminase polypeptides to carryout the conversion of large prochiral ketone substrate compounds, otherthan compound (2), to the chiral amine compounds, other than compound(1). In some embodiments, the engineered transaminase polypeptides ofthe present disclosure are capable of converting large prochiral ketonesubstrate compounds which are structural analogs of compound (2), to thecorresponding chiral amine product compounds which are structuralanalogs of compound (1). The range of large ketone substrate structuralanalog compounds capable of undergoing catalytic conversion using theengineered transaminase polypeptides provided by the present disclosureis illustrated by the conversion of compound of Formula (II) to thecompound of Formula (I) shown in Scheme 4.

As shown in Scheme 4, the large substrate ketone compound of Formula(II) has a structure comprising four rings with the prochiral ketonegroup that is converted to a chiral amine located at position 1 of ringA. Rings A and B are 6-membered carbocyclic rings optionally substitutedindependently at one or more of positions 2-10; ring C is a 5- or6-membered carbocyclic ring (i.e., m=0 or 1), optionally substituted atposition 11; and ring D is a 5-, 6-, or 7-membered carbocyclic ring(i.e., n=0, 1, or 2), optionally substituted independently at positions14, 15, and 16. The structural features of the engineered transaminasepolypeptides of the disclosure are capable of accommodating substratescompounds of Formula (II) that have large groups substituted atpositions 14, 15, and 16 of ring D while maintaining activity in thestereoselective conversion of the ketone at position 1 of ring A of thecompound of Formula (II) to a chiral amine. Without being bound bytheory, the structure of the engineered transaminase polypeptides of thedisclosure allows large groups substituted at positions 14, 15, and 16of ring D to extend into the solvent surrounding the enzyme, whilemaintaining the ketone at the 1 position of ring A in the appropriateposition of the active site for stereoselective transamination.Additionally, the binding pocket of the engineered transaminasepolypeptides of the present disclosure allows for substitutions ofsmaller groups at certain positions on rings A, B, and C (as describedfurther below), while maintaining activity in the stereoselectiveconversion of the ketone at position 1 of ring A of the compound ofFormula (II) to a chiral amine

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting the ketone substrate compounds ofFormula (II) to the corresponding chiral amine compounds of Formula (I)wherein rings A-D of the compounds can be substituted as follows:

Ring A is a 6-membered carbocyclic ring, optionally including anunsaturated C—C bond between positions 2 and 3 and/or positions 5 and 6,and/or optionally substituted independently positions 2, 3, 4, 5 and 6with a group selected from halo, hydroxy, and methyl;

Ring B is a 6-membered carbocyclic ring, optionally including anunsaturated C—C bond between positions 5 and 10, and/or optionallysubstituted independently at one or more of positions 9 and 10 with agroup selected from halo, hydroxy, and methyl;

Ring C is a 5- or 6-membered carbocyclic ring (i.e., m=0 or 1),optionally substituted at position 10 with a group selected from halo,hydroxy, methyl, ethyl, and carbonyl;

Ring D is a 5-, 6-, or 7-membered carbocyclic ring (i.e., n=0, 1, or 2),optionally including 1, 2, or 3 unsaturated C—C bonds, and/or optionallysubstituted independently as follows:

-   -   at position 14 with a group selected from halo, hydroxy, amino,        carboxy, cyano, nitro, thio, straight-chain or branched        (C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl,        straight-chain or branched (C₁-C₃)alkylamino, and cyclopropyl        bridging to position 12;    -   at position 15 or position 16 with a group selected from halo,        hydroxy, amino, carboxy, cyano, nitro, thio, optionally        substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, optionally        substituted(C₁-C₆)alkyloxy, optionally substituted        (C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,        optionally substituted (C₁-C₆)alkylthio, optionally substituted        (C₁-C₆)alkylsulfonyl, optionally substituted        (C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl,        (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy, optionally        substituted aminocarbonyl, aminocarbonyl(C₁-C₆)alkyl, optionally        substituted cycloalkyl, optionally substituted heterocycloalkyl,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted aryloxy, optionally substituted        arylamino, optionally substituted arylthio, optionally        substituted arylsulfonyl, optionally substituted arylsulfinyl,        optionally substituted aryloxycarbonyl, optionally substituted        arylcarbonyloxy, optionally substituted heteroaryloxy,        optionally substituted heteroarylamino, optionally substituted        heteroarylthio, optionally substituted heteroarylsulfonyl,        optionally substituted heteroarylsulfinyl, optionally        substituted heteroaryloxycarbonyl, optionally substituted        heteroarylcarbonyloxy, alkylaminosulfonyl(C₁-C₆)alkyl,        arylsulfonyl(C₁-C₆)alkyl, and heteroarylsulfonyl(C₁-C₆)alkyl.

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting ketone substrate compounds ofFormula (II) that are cyclopamine analog compounds such as the compoundsof Formula (IIa), wherein Ring C is a 5-membered carbocyclic ring,optionally substituted at position 11, and Ring D is a 7-memberedcarbocyclic ring substituted at position 16, which can be converted tothe chiral amine product of Formula (Ia) as shown in Scheme 5:

wherein

Rings A and B comprise one of the following:

-   -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D comprises an unsaturated C—C bond between positions 12 and 14;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl.

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting ketone substrate compounds ofFormula (II) that are cyclopamine analog compounds such as the compoundsof Formula (IIb), wherein Ring C is 5-membered carbocyclic ring and RingD is a 6-membered carbocyclic ring, which can be converted to the chiralamine product of Formula (Ib) as shown in Scheme 6:

wherein

Rings A and B comprise one of the following:

-   -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D comprises an unsaturated C—C bond between positions 12 and 14, ora bridging cyclopropyl between positions 12 and 14;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl.

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting ketone substrate compounds ofFormula (II) that are veratramine analog compounds such as the compoundsof Formula (IIc), wherein Ring C is 5-membered carbocyclic ring and RingD is a 6-membered carbocyclic ring, which can be converted to the chiralamine product of Formula (Ic) as shown in Scheme 7:

wherein

Rings A and B comprise one of the following:

-   -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D is aromatic;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl.

In some embodiments, In some embodiments, the engineered transaminasepolypeptides of the disclosure are capable of converting ketonesubstrate compounds of Formula (II) that are steroid analog compoundssuch as the compounds of Formula (IId), wherein Ring C is 6-memberedcarbocyclic ring and Ring D is a 5-membered carbocyclic ring, which canbe converted to the chiral amine product of Formula (Id) as shown inScheme 8:

wherein

Ring A comprises an unsaturated C—C bond between positions 2 and 3, orpositions 5 and 6;

R¹ and R² are selected independently from hydrogen, halo, hydroxy,amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionallysubstituted (C₁-C₆)alkylamino, optionally substituted(C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionallysubstituted (C₁-C₆)alkylsulfonyl, optionally substituted(C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl,(C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl,aminocarbonyl(C₁-C₆)alkyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryloxy, optionallysubstituted arylamino, optionally substituted arylthio, optionallysubstituted arylsulfonyl, optionally substituted arylsulfinyl,optionally substituted aryloxycarbonyl, optionally substitutedarylcarbonyloxy, optionally substituted heteroaryloxy, optionallysubstituted heteroarylamino, optionally substituted heteroarylthio,optionally substituted heteroarylsulfonyl, optionally substitutedheteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl,optionally substituted heteroarylcarbonyloxy,alkylaminosulfonyl(C₁-C₆)alkyl, arylsulfonyl(C₁-C₆)alkyl, andheteroarylsulfonyl(C₁-C₆)alkyl;

R³, R⁴, and R⁵ are selected independently from hydrogen, halo, hydroxy,amino, carboxy, cyano, nitro, thio, straight-chain or branched(C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, andstraight-chain or branched (C₁-C₃)alkylamino; and

R⁶, R⁷, and R⁸ are selected independently from hydrogen, halo, hydroxy,and methyl.

The engineered transaminase polypeptides adapted for efficientconversion of large ketone substrate compounds of Formula (II) to chiralamine product compounds of Formula (I) have one or more residuedifferences as compared to the amino acid sequence of the referenceengineered transaminase polypeptide of SEQ ID NO: 2. The residuedifferences are associated with enhancements in enzyme properties,including enzymatic activity, enzyme stability, and resistance toinhibition by the product amine

In some embodiments, the engineered transaminase polypeptides showincreased activity in the conversion of substrate compounds of Formula(II) (e.g., compound (2)) to the amino product compounds of Formula (I)(e.g., compound (1)) in diastereomeric excess in a defined time with thesame amount of enzyme as compared to the wild-type or the referenceengineered transaminase of SEQ ID NO: 4. In some embodiments, theengineered transaminase polypeptide has at least about 1.2 fold, 1.5fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40fold, or 50 fold or more the activity as compared to the referenceengineered polypeptide represented by SEQ ID NO:4 under suitablereaction conditions.

In some embodiments, the engineered transaminase polypeptides haveincreased stability to temperature and/or solvents used in theconversion reaction as compared to the wild-type or a referenceengineered enzyme. In some embodiments, the engineered transaminasepolypeptide has at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5fold, 10 fold or more the stability as compared to the referencepolypeptide of SEQ ID NO: 4 under suitable reaction conditions.

In some embodiments, the engineered transaminase polypeptides haveincreased refractoriness or resistance to inhibition by product chiralamine of compound (1) as compared to the wild-type or a referenceengineered enzyme. In some embodiments, the engineered transaminasepolypeptide has at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5fold, or more increased resistance to inhibition by the product ofcompound (1), as compared to the polypeptide represented by SEQ ID NO:4under suitable reaction conditions, as further described below.

In some embodiments, the engineered transaminase polypeptides arecapable of converting the substrate of compound (2) to compound (1) indiastereomeric excess of greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5% or greater under suitable reaction conditions(i.e., excess over other diastereomeric product compounds having the theopposite enantiomer at the chiral amine center).

In some embodiments, the engineered transaminase polypeptides arecapable of converting substrate compound (2) to product compound (1)with increased tolerance for the presence of substrate relative to thereference polypeptide of SEQ ID NO: 4 under suitable reactionconditions. Thus, in some embodiments the engineered transaminasepolypeptides are capable of converting the substrate compound (2) toproduct compound (1) under a substrate loading concentration of at leastabout 1 g/L, about 5 g/L, about 10 g/L, about 20 g/L, about 30 g/L,about 40 g/L, about 50 g/L, about 70 g/L, about 100 g/L, about 125 g/L,about 150 g/L, about 175 g/L or about 200 g/L or more with a percentconversion of at least about at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 98%, or at least about 99%, in a reaction time ofabout 72 h or less, about 48 h or less, about 36 h or less, or about 24h less, under suitable reaction conditions.

The suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides carry out theconversion can be determined with respect to concentrations or amountsof polypeptide, substrate, cofactor, buffer, co-solvent, pH, and/orconditions including temperature and reaction time, as further describedbelow and in the Examples.

The present disclosure provides 200 exemplary engineered transaminasepolypeptides having structural features capable of converting largeprochiral ketone substrate compounds of Formula (II), which arestructural analogs of compound (2), to the corresponding chiral amineproduct compounds of Formula (I), which are structural analogs ofcompound (1). The present disclosure provides the sequence structure ofthe 200 exemplary engineered transaminase polypeptides as SEQ ID NOs:5-204 in the electronic Sequence Listing file accompanying thisdisclosure, which is hereby incorporated by reference herein. The oddnumbered sequence identifiers (i.e., SEQ ID NOs) refer to the nucleotidesequence encoding the amino acid sequence provided by the even numberedSEQ ID NOs. The present disclosure also provides in Tables 2A and 2Bsequence structural information correlating specific amino acid sequencefeatures with the functional activity of the engineered transaminasepolypeptides. This structure-function correlation information isprovided in the form of specific amino acid residues differencesrelative to the reference engineered polypeptide of SEQ ID NO: 2 andassociated experimentally determined activity data for the 200 exemplaryengineered transaminases of SEQ ID NOs: 5-204. The amino acid residuedifferences are based on comparison to the reference sequence of SEQ IDNO: 2, which has the following 10 amino acid residue differencesrelative to the sequence of the wild-type ω-VfT polypeptide (Accession:gi|327207066|gb|AEA39183.1|): A9T; N45H; W57L; F86S; V153A; V177L;R211K; M294V; S324G; and T391A. The relative transaminase activity ofeach exemplary engineered transaminase polypeptide was determined asconversion of the prototype large substrate ketone of compound (2), tothe chiral amine product of compound (1) in comparison to thetransaminase activity of the engineered transaminase polypeptide of SEQID NO: 4 over a set time period and temperature in a high-throughput(HTP) assay, which was used as the primary screen. The engineeredtransaminase polypeptide of SEQ ID NO: 4 used as the activity referencehas the following 8 amino acid residue differences relative to thereference sequence of SEQ ID NO: 2: T34A; L56A; R88H; A153C; A155V;K163F; E315G; and L417T. The HTP Activity assay values in Table 2A weredetermined using E. coli. clear cell lysates in 96 well-plate format of˜200 μL volume per well following assay reaction conditions as noted inthe table and the Examples.

TABLE 2A Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ HTP Activity¹ ID NO: Amino Acid Differences(relative to (nt/aa) (relative to SEQ ID NO: 2) SEQ ID NO: 4) % de 3/4T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1 98.6 L417T; 5/6 T34A;N53M; L56A; S86C; R88Y; R146L; A153C; A155V; 1.57 n.d. K163F; Y165F;E315G; R366H; A383V; L417T 7/8 T34A; N53M; L56A; S86C; R88Y; R146L;A153C; A155V; 2.19 n.d. K163F; Y165F; A228G; I259V; E315G; R366H; A383V;L417T  9/10 T34A; N53M; L56A; S86C; R88Y; R146L; A153C; A155V; 1.37 n.d.K163F; Y165F; I259V; E315G; R366H; A383V; R415A; L417T 11/12 T34A; N53M;L56A; S86C; R88Y; R146L; A153C; A155V; 1.40 n.d. K163F; Y165F; I259V;E315G; A383V; R415A; L417T 13/14 T34A; N53M; L56A; S86C; R88Y; R146L;A153C; A155V; 2.30 n.d. K163F; Y165F; A228G; I259V; E315G; R366H; A383V;R415V; L417T 15/16 T34A; N53M; L56A; S86C; R88Y; R146L; A153C; A155V;2.31 n.d. K163F; Y165F; A228G; I259V; E315G; R366H; A383V; R415A; L417T17/18 T34A; N53M; L56A; S86C; R88Y; R146L; A153C; A155V; 1.67 n.d.K163F; Y165F; I259V; E315G; R366H; A383V; R415G; L417T 19/20 T34A; N53M;S86C; R88Y; R146L; A153C; A155V; 1.63 n.d. K163F; Y165F; A228G; E315G;R366H; A383V; L417T 21/22 T34A; N53M; S86C; R88Y; R146L; A153C; A155V;1.90 n.d. K163F; Y165F; I259V; E315G; R366H; A383V; R415V; L417T 23/24T34A; N53M; L56A; S86C; R88Y; R146L; A153C; A155V; 2.25 n.d. K163F;Y165F; A228G; I259V; T277A; E315G; R366H; A383V; R415G; L417T 25/26T34A; N53M; L56A; S86C; R88Y; R146L; A153C; A155V; 2.71 n.d. K163F;Y165F; I259V; E315G; R366H; A383V; L417T 27/28 T34A; N53M; L56A; S86C;R88Y; R146L; A153C; A155V; 1.46 n.d. K163F; Y165F; A228G; I251V; I259V;E315G; R366H; A383V; V399A; L417T 29/30 G18A; T34A; L56A; R88H; A153C;A155V; K163F; 2.11 n.d. P233T; E315G; A383V; L417T 31/32 V31M; T34A;L56A; R88H; A153C; A155V; K163F; 1.57 n.d. P233T; P244T; E315G; A383V;L417T; 33/34 V31M; T34A; L56A; R88H; A153C; A155V; K163F; 2.23 n.d.E315G; A383V; L417T; C424A; 35/36 D21H; V31M; T34A; L56A; R88H; A153C;A155V; 1.99 n.d. K163F; P244T; E315G; A383V; L417T; 37/38 V31M; T34A;L56A; R88H; A153C; A155V; K163F; 2.32 n.d. E315G; A383V; L417T; F427Y;39/40 V31M; T34A; L56A; R88H; A153C; A155V; K163F; 2.42 n.d. P233T;E315G; A383V; L417T; C424A; 41/42 D21H; V31M; T34A; L56A; R88H; R146L;A153C; 1.79 n.d. A155V; K163F; P233T; E315G; A383V; L417T; 43/44 V31M;T34A; L56A; R88H; A153C; A155V; K163F; 2.21 n.d. E315G; A383V; L417T;45/46 V31M; T34A; L56A; R88H; A153C; A155V; K163F; 1.72 n.d. P244T;E315G; A383V; L417T; 47/48 V31M; T34A; L56A; R88H; R146L; A153C; A155V;2.00 n.d. K163F; P233T; A235P; P244T; E315G; A383V; L417T; C424A; F427Y;49/50 V31M; T34A; L56A; R88H; A153C; A155V; K163F; 2.23 n.d. E315G;A383V; L417T; C424A; F427Y; 51/52 V31M; T34A; L56A; R88H; A153C; A155V;K163F; 2.40 n.d. P233T; E315G; A383V; L417T; F427Y; 53/54 V31M; T34A;L56A; R88H; A153C; A155V; K163F; 1.58 n.d. P233T; P244T; E315G; A383V;L417T; F427Y; 55/56 V31M; T34A; L56A; R88H; A153C; A155V; K163F; 1.40n.d. P233T; E315G; L417T; C424A; 57/58 T34A; L56A; R88H; A153C; A155V;K163F; A383V; 1.69 n.d. E315G; L417T; 59/60 V31M; T34A; L56A; R88H;A153C; A155V; K163F; 1.94 n.d. W147K; P233T; P244T; E315G; A383V; L417T;61/62 V31M; T34A; L56A; R88H; A153C; A155V; K163F; 1.44 n.d. E315G;L417T; C424A; 63/64 D21H; V31M; T34A; L56A; R88H; A153C; A155V; 1.48n.d. K163F; E315G; L417T; 65/66 V31M; T34A; L56A; R88H; R146L; A153C;A155V; 1.62 n.d. K163F; E315G; A383V; L417T; F427Y; 67/68 G18A; V31M;T34A; L56A; R88H; A153C; A155V; 2.33 n.d. K163F; P233T; E315G; A383V;L417T; C424A; 69/70 D21H; V31M; T34A; L56A; R88H; A153C; A155V; 2.61n.d. K163F; E315G; A383V; L417T; F427Y; 71/72 F19W; T34A; L56A; R88H;A153C; A155V; K163F; 0.24 n.d. E315G; E358K; L417T; 73/74 T34A; L56C;R88H; A153C; A155V; K163F; E315G; 0.67 n.d. L417T; 75/76 T34A; L56A;L57F; R88H; A153C; A155V; K163F; 0.93 n.d. E315G; L417T; 77/78 T34A;L56A; L57C; R88H; A153C; A155V; K163F; 1.30 n.d. E315G; L417T; 79/80T34A; L56A; S86N; R88H; A153C; A155V; K163F; 0.72 n.d. E315G; L417T;81/82 T34A; L56A; R88H; A153C; A155V; K163L; E315G; 1.38 n.d. A323T;L417T; M434T; 83/84 T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1.39n.d. R415L; L417T; 85/86 T34A; L56A; R88H; A153C; A155V; K163F; E315G;1.31 n.d. R415H; L417T; 87/88 T34A; L56A; R88H; A153C; A155V; K163F;T268A; 1.71 n.d. E315G; A383F; L417T; 89/90 T34A; L56A; R88H; A153C;A155V; K163F; N286H; 1.00 n.d. E315G; L417T; 91/92 T34A; L56A; R88H;A153C; A155V; K163F; E315G; 1.85 n.d. E316S; L417T; 93/94 T34A; L56A;R88H; A153C; A155V; K163F; E315G; 1.61 n.d. E316C; L417T; 95/96 T34A;L56A; R88H; A153C; A155V; K163F; E315G; 1.16 n.d. G395P; L417T; 97/98T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1.67 n.d. E316T; L417T; 99/100 T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1.88 n.d. E316N;L417T; 101/102 T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1.73 n.d.E316F; L417T; 103/104 T34A; L56A; R88H; A153C; A155V; K163F; N286C; 1.56n.d. E315G; L417T; 105/106 T34A; L56A; R88H; D107G; A153C; A155V; K163F;1.28 n.d. E315G; L417T; 107/108 T34A; L56A; R88H; Y113P; A153C; A155V;K163F; 1.51 n.d. E315G; L417T; 109/110 T34A; L56A; R88H; Y113L; A153C;A155V; K163F; 1.58 n.d. E315G; L417T; 111/112 T34A; L56A; R88H; Y113C;A153C; A155V; K163F; 1.71 n.d. E315G; L417T; 113/114 T34A; L56A; R88H;W147V; A153C; A155V; K163F; 1.49 n.d. E315G; L417T; 115/116 T34A; L56A;R88H; W147H; A153C; A155V; K163F; 1.54 n.d. E315G; L417T; 117/118 T34A;L56A; R88H; A153C; A155V; K163F; H178W; 1.12 n.d. E315G; L417T; 119/120T34A; L56A; R88H; A153C; A155V; K163F; P233V; 1.35 n.d. E315G; L417T;121/122 T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1.81 n.d. A323T;L417T; 123/124 T34A; L56A; R88H; A153C; A155V; K163F; E315G; 2.67 n.d.A383T; L417T; 125/126 T34A; L56A; R88H; A153C; A155V; K163F; E315G; 2.56n.d. C414I; L417T; 127/128 T34A; L56A; R88H; A153C; A155V; K163F; P233T;2.67 n.d. E315G; L417T; 129/130 T34A; L56A; R88H; A153C; A155V; K163F;E315G; 1.43 n.d. A383C; L417T; 131/132 T34A; L56A; R88H; A153C; A155V;K163F; E315G; 2.50 n.d. A383I; L417T; 133/134 T34A; L56A; R88H; A153C;A155V; K163F; E315G; 2.61 n.d. L417T; A450S; 135/136 T34A; L56A; R88H;A153C; A155V; K163F; E206K; 1.44 n.d. E315G; E316A; L417T; 137/138 T34A;L56A; R88H; A153C; A155V; K163F; E315G; 1.72 n.d. A383F; L417T; 139/140T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1.77 n.d. A383M; L417T;141/142 T34A; L56A; R88H; A153C; A155V; K163F; E315G; 1.40 n.d. L417V;143/144 T34A; L56A; K73R; R88H; A153C; A155V; K163F; 1.34 n.d. E315G;A383L; L417T; 145/146 V31M; T34A; L56A; L57F; R88H; A153C; A155V; K163F;5.61 95.4 N286C; E315G; E316N; A383V; R415H; L417T; 147/148 V31M; T34A;L56A; L57F; R88H; A153C; A155V; K163F; 5.79 99.4 E315G; E316N; A323T;A383V; L417T; 149/150 V31M; T34A; L56A; L57F; R88H; Y113C; W147V; 7.8293.1 A153C; A155V; K163F; N286C; E315G; E316S; A323T; A383M; R415H;L417T; A450S; 151/152 V31M; T34A; L56A; L57F; R88H; Y113L; A153C; A155V;5.96 94.8 K163F; E190K; P233V; E315G; E316N; A383M; R415H; L417T; A450S;153/154 V31M; T34A; L56A; L57F; R88H; D107G; Y113L; 5.84 94.1 W147V;A153C; A155V; K163F; P233V; E315G; E316N; A383T; R415H; L417T; A450S;155/156 V31M; T34A; L56A; L57F; R88H; D107G; Y113L; A153C; 6.07 99.4A155V; K163F; P233T; E315G; E316N; A323T; L417T; 157/158 V31M; T34A;L56A; L57F; R88H; D107G; W147V; 7.61 94.6 A153C; A155V; K163F; E315G;E316N; A323T; R415H; L417T; 159/160 V31M; T34A; L56A; L57F; R88H; A153C;A155V; K163F; 8.85 93.9 P233V; E315G; E316N; A323T; A383I; R415H; L417T;A450S; 161/162 V31M; T34A; L56A; L57F; R88H; W147V; A153C; 5.75 98.9A155V; K163F; N286C; E315G; E316N; A323T; A383I; C414I; L417T; A450S;163/164 V31M; T34A; L56A; L57F; R88H; A153C; A155V; K163F; 5.42 98.9P233T; N286C; E315G; E316N; A323T; A383I; L417T; A450S; 165/166 V31M;T34A; L56A; L57F; R88H; W147V; A153C; 7.41 93.7 A155V; K163F; P233V;E315G; E316N; A323T; A383T; R415H; L417T; A450S; 167/168 V31M; T34A;L56A; L57F; R88H; A153C; A155V; K163F; 5.81 95.6 E315G; E316N; A383I;R415H; L417T; 169/170 V31M; T34A; L56A; L57F; R88H; A153C; A155V; K163F;6.04 98.5 N286C; E315G; E316S; A323T; A383I; L417T; 171/172 V31M; T34A;L56A; L57F; R88H; A153C; A155V; K163F; 5.68 98.7 P233V; E315G; E316S;A323T; A383V; L417T; 173/174 V31M; T34A; L56A; L57F; R88H; Y113C; A153C;A155V; 5.23 99.4 K163F; P233V; E315G; E316N; A383I; L417T; A450S;175/176 T34A; L56A; L57F; R88H; W147H; A153C; A155V; 6.80 97.0 K163F;N286C; E315G; E316S; A323T; R415H; L417T; 177/178 V31M; T34A; L56A;L57F; R88H; A153C; A155V; K163F; 5.08 99.4 N286C; E316N; A323T; A450S;E315G; L417T; 179/180 V31M; T34A; L56A; L57F; R88H; W147H; A153C; 6.2299.0 A155V; K163F; E315G; E316N; A323T; A383I; L417T; A450S; 181/182V31M; L57F; W147H; P233V; E316S; A323T; A383I; 5.62 98.8 A450S; T34A;L56A; R88H; A153C; A155V; K163F; E315G; L417T; 183/184 V31M; T34A; L56A;L57F; R88H; A153C; A155V; K163F; 5.39 96.4 P233V; N286C; E315G; E316N;H319N; A323T; A383T; R415H; L417T; 185/186 V31M; T34A; L56A; R88H; L57F;Y113C; A153C; A155V; 5.70 96.7 K163F; E315G; E316N; R415H; L417T;187/188 V31M; T34A; L56A; L57F; R88H; W147H; A153C; 5.38 99.4 A155V;K163F; E315G; E316N; A383I; L417T; 189/190 V31M; T34A; L56A; L57F; R88H;W147V; A153C; 5.17 99.3 A155V; K163F; P233V; N286C; E315G; E316N; A323T;A383M; L417T; A450S; 191/192 V31M; T34A; L56A; L57F; R88H; A153C; A155V;K163F; 5.58 95.5 P233V; E315G; E316N; F317L; A323T; A383V; R415H; L417T;193/194 V31M; T34A; N53M; L56A; L57F; S86C; R88Y; R146L; 5.04 95.9A153C; A155V; K163F; Y165F; I259V; E315G; R366H; A383V; L417T; C424A;195/196 T34A; N53M; L56A; L57F; S86C; R88Y; R146L; A153V; 5.11 99.0A155V; K163F; Y165F; I259V; E312N; I314N; E315G; R366H; A383V; L417T;C424A; 197/198 T34A; N53M; L56A; L57F; S86C; R88Y; R146L; A153C; 6.3898.6 A155V; K163F; Y165F; I259V; E315G; R366H; A383V; L417T; C424A;199/200 V31M; T34A; N53M; L56A; L57F; S86C; R88Y; R146L; 4.85 95.4A153V; A155V; K163F; Y165F; L171Q; I259V; E315G; R366H; A383V; L417T;C424A; P426R; 201/202 T34A; N53M; L56A; S86C; R88Y; R146L; A153C; A155V;5.59 95.8 K163F; Y165F; I259V; E312N; E315G; E316G; R366H; A383V; L417T;C424A; 203/204 V31M; T34A; N53M; L56A; L57F; S86C; R88Y; R146L; 5.4095.7 A153C; A155V; K163F; Y165F; I259V; E312N; E315G; R366H; A383V;L417T; C424A; ¹HTP Activity Improvement (relative to SEQ ID NO: 4) iscalculated as the ratio of % conversion of product formed by theengineered transaminase polypeptide of interest to the % conversion ofthe reference polypeptide of SEQ ID NO: 4 under Reaction Conditions A. %Conversion was quantified by dividing the areas of the product peak bythe sum of the areas of the substrate and product peak as determined byHPLC analysis. Reaction Conditions A: 20 g/L substrate, 10 μL lysate(prepared by adding 200 μL of Lysis Buffer (1 mg/mL lysozyme, 0.5 mg/mLpolymyxin B sulfate, 1 mM PLP, 0.1M triethanolamine (TEA), pH 7.0) to E.coli expressing polypeptide of interest grown in 96 well plates), 0.5g/L pyridoxal-5′-phosphate (PLP), 1M isopropylamine (IPM), 25% DMSO, pH8.0, 60° C., 24 h. Total reaction volume is 200 μL. “n.d.” = notdetermined

In some instances, a shake-flask powder (SFP) and/or downstreamprocessed (DSP) powder assay were used as a secondary screen to assessthe properties of the exemplary engineered transaminase polypeptides,the results of which are provided in Table 2B. The SFP and DSP formsprovide a more purified powder preparation of the engineeredpolypeptides. For example, the engineered transaminase in a SFPpreparation is approximately 30% of the total protein in the preparationwhile the engineered transaminase in a DSP preparation is approximately80% of total protein. Assessment of stability was made by comparingactivities at two different temperatures, 55° C. and 60° C.

TABLE 2B Engineered Transaminase Polypeptides and Relative ImprovementsUsing Shake Flask and DSP Enzyme Preparations SEQ ID % % NO: Amino AcidDifferences Conversion % de Conversion % de (nt/aa) (relative to SEQ IDNO: 2) (24 h at 55° C.) (55° C.) (24 h at 60° C.) (60° C.) SFP enzymepreparation assayed using reaction conditions B¹ 3/4 T34A; L56A; R88H;A153C; 41.2 98.6 26.0 98.4 A155V; K163F; E315G; L417T 7/8 T34A; N53M;L56A; S86C; 96.2 85.8 84.8 83.5 R88Y; R146L; A153C; A155V; K163F; Y165F;A228G; I259V; E315G; R366H; A383V; L417T 25/26 T34A; N53M; L56A; S86C;95.6 92.0 98.0 91.6 R88Y; R146L; A153C; A155V; K163F; Y165F; I259V;E315G; R366H; A383V; L417T 35/36 D21H; V31M; T34A; L56A; 79.4 96.4 52.795.3 R88H; A153C; A155V; K163F; P244T; E315G; A383V; L417T; 39/40 V31M;T34A; L56A; R88H; 95.2 96.8 66.9 95.9 A153C; A155V; K163F; P233T; E315G;A383V; L417T; C424A; 77/78 T34A; L56A; L57C; R88H; 67.5 96.9 31.4 97.0A153C; A155V; K163F; E315G; L417T;  99/100 T34A; L56A; R88H; A153C; 86.998.4 23.1 100.0 A155V; K163F; E315G; E316N; L417T; 101/102 T34A; L56A;R88H; A153C; 81.1 97.5 20.1 100.0 A155V; K163F; E315G; E316F; L417T; SFPenzyme preparations assayed using reaction conditions C²  99/100 T34A;L56A; R88H; A153C; 49.8 100.0 13.0 100.0 A155V; K163F; E315G; E316N;L417T; 147/148 V31M; T34A; L56A; L57F; 93.9 100.0 94.1 100.0 R88H;A153C; A155V; K163F; E315G; E316N; A323T; A383V; L417T; 155/156 V31M;T34A; L56A; L57F; 93.7 99.3 75.4 100.0 R88H; D107G; Y113L; A153C; A155V;K163F; P233T; E315G; E316N; A323T; L417T; 159/160 V31M; T34A; L56A;L57F; 98.3 95.7 90.5 96.2 R88H; A153C; A155V; K163F; P233V; E315G;E316N; A323T; A383I; R415H; L417T; A450S; 169/170 V31M; T34A; L56A;L57F; 93.9 99.4 95.6 98.4 R88H; A153C; A155V; K163F; N286C; E315G;E316S; A323T; A383I; L417T; 171/172 V31M; T34A; L56A; L57F; 96.0 99.694.3 99.5 R88H; A153C; A155V; K163F; P233V; E315G; E316S; A323T; A383V;L417T; 179/180 V31M; T34A; L56A; L57F; 96.9 100.0 91.9 100.0 R88H;W147H; A153C; A155V; K163F; E315G; E316N; A323T; A383I; L417T; A450S;197/198 T34A; N53M; L56A; L57F; S86C; 48.1 97.2 82.0 98.0 R88Y; R146L;A153C; A155V; K163F; Y165F; I259V; E315G; R366H; A383V; L417T; C424A;DSP enzyme preparations assayed using reaction conditions D³ 3/4 T34A;L56A; R88H; A153C; 22.3 98.6 15.2 98.6 A155V; K163F; E315G; L417T 99/100 T34A; L56A; R88H; A153C; 49.3 >99 23.5 >99 A155V; K163F; E315G;E316N; L417T; 147/148 V31M; T34A; L56A; L57F; 96.1 >99 96.8 98.9 R88H;A153C; A155V; K163F; E315G; E316N; A323T; A383V; L417T; 155/156 V31M;T34A; L56A; L57F; 96.4 >99 96.7 >99 R88H; D107G; Y113L; A153C; A155V;K163F; P233T; E315G; E316N; A323T; L417T; 159/160 V31M; T34A; L56A;L57F; 96.1 97.3 97.0 95.7 R88H; A153C; A155V; K163F; P233V; E315G;E316N; A323T; A383I; R415H; L417T; A450S; 179/180 V31M; T34A; L56A;L57F; 96.3 98.8 94.3 99.2 R88H; W147H; A153C; A155V; K163F; E315G;E316N; A323T; A383I; L417T; A450S; ¹Reaction Conditions B: 20 g/Lsubstrate, 4 g/L SFP enzyme preparation, 0.5 g/L pyridoxal-5′-phosphate(PLP), 1M isopropylamine (IPM), 25% v/v DMSO, pH 8.0, 55° C. and 60° C.Total reaction volume: 10 mL. ²Reaction Conditions C: 20 g/L substrate,2 g/L SFP enzyme preparation, 0.5 g/L pyridoxal-5′-phosphate (PLP), 1Misopropylamine (IPM), 25% v/v DMSO, pH 8.0, 55° C. and 60° C. Totalreaction volume: 10 mL. Reaction Conditions D: 20 g/L substrate, 2 g/LDSP enzyme preparation, 0.5 g/L pyridoxal-5′-phosphate (PLP), 1Misopropylamine (IPM), 25% v/v DMSO, pH 8.0, 55° C. and 60° C. Totalreaction volume: 10 mL.

From an inspection of the amino acid sequences, and results for the 200exemplary engineered polypeptides of Tables 2A and 2B, improvedproperties of increased activity, enantioselectivity, and/or stability,that are associated with one or more residue differences as compared toSEQ ID NO:4 at the following residue positions: X18, X19, X21, X31, X34,X53, X56, X57 X73, X86, X88, X107, X113, X133, X147, X155, X163, X165,X171, X178, X190, X206, X228, X233, X235, X244, X251, X259, X268, X277,X286, X312, X314, X316, X317, X319, X323, X358, X366, X383, X395, X399,X414, X415, X417, X424, X426, X427, X434, and X450. The specific aminoacid differences at each of these positions that are associated with theimproved properties include: X18A; X19W; X21H; X31M; X53M; X56A/C;X57C/F; X73R; X86C/N; X88H/Y; X107G; X113C/L/P; X146L; X147H/KN; X153V;X155A; X163L; X165F; X171Q; X178W; X190K; X206K; X228G; X233T/V; X235P;X244T; X251V; X259V; X268A; X277A; X286C/H; X312N; X314N;X316A/C/F/N/S/T; X317L; X319N; X323T; X358K; X366H; X383C/F/I/L/M/T/V;X395P; X399A; X414I; X415A/G/H/L/V; X417V; X424A; X426R; X427Y; X434T;and X450S.

In some embodiments, the engineered transaminase polypeptides of thepresent disclosure comprise amino acid sequences having residuedifferences as compared to the engineered transaminase represented bySEQ ID NO:4 at residue positions selected from: X19, X21, X34, X53, X56,X73, X86, X88, X107, X113, X133, X147, X155, X165, X171, X178, X233,X251, X259, X268, X277, X286, X312, X316, X317, X323, X358, X366, X383,X399, X414, X415, X417, X426, X434, and X450, wherein the residuedifferences at residue positions X21, X56, X86, X88, X107, X113, X133,X147, X233, X286, X312, X316, X323, X383, X415, X417, and X434, areselected from: X21H, X56A/C, X86C, X88H/Y, X107G, X113L/P, X133A,X147H/V, X233V, X286C/H, X312N, X316C/F/G/N/S/T, X323A, X383C/F/I/M/T,X415A/G/H/L/V, X417V, and X434T.

In some embodiments, the engineered transaminase polypeptides of thepresent disclosure comprise amino acid sequences having residuedifferences as compared to the engineered transaminase represented bySEQ ID NO:4 at residue positions selected from: X19, X34, X53, X73,X155, X165, X171, X178, X251, X259, X268, X277, X317, X358, X366, X399,X414, X426, and X450. In some embodiments, the specific amino aciddifferences at positions X19, X34, X53, X73, X155, X165, X171, X178,X251, X259, X268, X277, X317, X358, X366, X399, X414, X426, and X450 areselected from: X19W, X34A, X53M, X73R, X155V, X165F, X171Q, X178W,X251V, X259V, X268A, X277A, X317L, X358K, X366H, X399A, X414I, X426R,and X450S.

The specific enzyme properties associated with the residues differencesas compared to SEQ ID NO:4 at the residue positions above include, amongothers, enzyme activity, and stability. Residue differences associatedwith increased enzyme stability are associated with residue differencesat residue positions X34, X107, X113, X147, X155, X233, X323, X383, andX450, including the specific residue differences, X34T, X107G, X113L,X147H, X155V, X233T/V, X323T, X383I/V, and X450S. Residue differencesassociated with increased activity in the conversion of large ketonesubstrates of Formula (II) to the corresponding chiral amine compound ofFormula (I) are associated with residue differences at residue positionsX56, X57, X86, X88, X153, X316, X415, and X417, including the specificresidue differences, X56A, X57F, X88H, X153C, X316N, X415H, and X417T.Residue differences specifically associated with increased % ee for theconversion of compounds of Formula (II), such as compound (2), tocompounds of Formula (I), such as compound (1), include X57F, X153C, andX316N.

As will be appreciated by the skilled artisan, residue differencesdisclosed in Tables 2A and 2B have no significant deleterious effects onthe activity and/or enantioselectivity of the engineered transaminasepolypeptides, which are maintain transaminase activity andenantioselectivity (85% d.e. or greater) for the conversion of compound(2) to compound (1). Nearly all of the polypeptides haveenantioselectivities equal to or greater than 95% de. Accordingly, theskilled artisan will understand that the residue differences at theresidue positions disclosed herein can be used individually or invarious combinations to produce engineered transaminase polypeptideshaving the desired functional properties, including, among others,transaminase activity, stereoselectivity, and stability, in convertinglarge ketone substrate compounds of Formula (II) to chiral aminecompounds of Formula (I).

In light of the guidance provided herein, it is further contemplatedthat any of the exemplary engineered polypeptides of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, and 204 can be used as the starting amino acidsequence for synthesizing other engineered transaminase polypeptides,for example by subsequent rounds of evolution by adding new combinationsof various amino acid differences from other polypeptides in Tables 2Aand 2B, and other residue positions described herein. Furtherimprovements may be generated by including amino acid differences atresidue positions that had been maintained as unchanged throughoutearlier rounds of evolution.

Accordingly, in some embodiments, the present disclosure providesengineered polypeptides having transaminase activity, and optionallyimproved properties in converting a ketone substrate compound (2) to achiral amine product compound (1) as compared to a reference polypeptideof SEQ ID NO:4, wherein the polypeptide comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequenceSEQ ID NO: 2 and one or more residue differences as compared to SEQ IDNO:2 at residue positions selected from X19, X21, X34, X53, X56, X73,X86, X88, X107, X113, X133, X147, X155, X165, X171, X178, X233, X251,X259, X268, X277, X286, X312, X316, X317, X323, X358, X366, X383, X399,X414, X415, X417, X426, X434, and X450, wherein the residue differencesat residue positions X21, X56, X86, X88, X107, X113, X133, X147, X233,X286, X312, X316, X323, X383, X415, X417, and X434, are selected from:X21H, X56A/C, X86C, X88H/Y, X107G, X113L/P, X133A, X147H/V, X233V,X286C/H, X312N, X316C/F/G/N/S/T, X323A, X383C/F/I/M/T, X415A/G/H/L/V,X417V, and X434T. In some embodiments, the specific amino aciddifferences at positions X19, X34, X53, X73, X155, X165, X171, X178,X251, X259, X268, X277, X317, X358, X366, X399, X414, X426, and X450 areselected from: X19W, X34A, X53M, X73R, X155V, X165F, X171Q, X178W,X251V, X259V, X268A, X277A, X317L, X358K, X366H, X399A, X414I, X426R,and X450S. In some embodiments, the engineered transaminase polypeptidesare capable converting substrate compound (2) to product compound (1)with the improved enantioselectivities described herein, e.g., ≥90% de.

In some embodiments, the engineered polypeptide having transaminaseactivity of the present disclosure comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204, and oneor more residue differences as compared to SEQ ID NO:2 at residuepositions selected from X19, X21, X34, X53, X56, X73, X86, X88, X107,X113, X133, X147, X155, X165, X171, X178, X233, X251, X259, X268, X277,X286, X312, X316, X317, X323, X358, X366, X383, X399, X414, X415, X417,X426, X434, and X450, wherein the residue differences at residuepositions X21, X56, X86, X88, X107, X113, X133, X147, X233, X286, X312,X316, X323, X383, X415, X417, and X434, are selected from: X21H, X56A/C,X86C, X88H/Y, X107G, X113L/P, X133A, X147H/V, X233V, X286C/H, X312N,X316C/F/G/N/S/T, X323A, X383C/F/I/M/T, X415A/G/H/LN, X417V, and X434T.In some embodiments, the specific amino acid differences at positionsX19, X34, X53, X73, X155, X165, X171, X178, X251, X259, X268, X277,X317, X358, X366, X399, X414, X426, and X450 are selected from: X19W,X34A, X53M, X73R, X155V, X165F, X171Q, X178W, X251V, X259V, X268A,X277A, X317L, X358K, X366H, X399A, X414I, X426R, and X450S. In someembodiments, the reference sequence is selected from SEQ ID NO: 4, 8,26, 36, 40, 78, 100, 102, 148, 156, 160, 170, 172, 180, and 198. In someembodiments, the reference sequence is SEQ ID NO:4. In some embodiments,the reference sequence is SEQ ID NO:100. In some embodiments, thereference sequence is SEQ ID NO:148. In some embodiments, the referencesequence is SEQ ID NO:156. In some embodiments, the reference sequenceis SEQ ID NO:160. In some embodiments, the reference sequence is SEQ IDNO:180.

In some embodiments, the engineered polypeptide having transaminaseactivity of the present disclosure comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204, and atleast the following combination of residue differences as compared toSEQ ID NO: 2 of X34A, X56A, X57L, X86S, X88A; X153C, X155V, X163F,X315G, and X417T. In some embodiments, the engineered polypeptide havingtransaminase activity further comprises a combination of residuedifferences selected from: (a) X31M, X57F, X316N, X323T, and X383V; (b)X31M, X57F, X107G, X113L, X233T, X316N, X415H, and X450S; (c) X31M,X57F, X233V, X316N, X323T, X383I, X415H, and X450S; and (d) X31M, X57F,X147H, X316N, X323T, X383I, X415H, and X450S.

As will be appreciated by the skilled artisan, in some embodiments, oneor a combination of residue differences above that is selected can beconserved in the engineered transaminases as a core sequence (orfeature), and additional residue differences at other residue positionsincorporated into the core sequence to generate additional engineeredtransaminase polypeptides with improved properties. Accordingly, it isto be understood for any engineered transaminase containing one or asubset of the residue differences above, the present disclosurecontemplates other engineered transaminases that comprise the one orsubset of the residue differences, and additionally one or more residuedifferences at the other residue positions disclosed herein. By way ofexample and not limitation, an engineered transaminase comprising aresidue difference at residue position X316, can further incorporate oneor more residue differences at the other residue positions, e.g., X19,X21, X34, X53, X56, X73, X86, X88, X107, X113, X133, X147, X155, X165,X171, X178, X233, X251, X259, X268, X277, X286, X312, X317, X323, X358,X366, X383, X399, X414, X415, X417, X426, X434, and X450. Anotherexample is an engineered transaminase comprising a residue difference atresidue position X56, which can further comprise one or more residuedifferences at the other residue positions, e.g., X19, X21, X34, X53,X73, X86, X88, X107, X113, X133, X147, X155, X165, X171, X178, X233,X251, X259, X268, X277, X286, X312, X316, X317, X323, X358, X366, X383,X399, X414, X415, X417, X426, X434, and X450. For each of the foregoingembodiments, the engineered transaminase can further comprise additionalresidue differences selected from: X18A; X19W; X21H; X31M; X53M; X56A/C;X57C/F; X73R; X86C/N; X88H/Y; X107G; X113C/L/P; X146L; X147H/K/V; X153V;X155A; X163L; X165F; X171Q; X178W; X190K; X206K; X228G; X233T/V; X235P;X244T; X251V; X259V; X268A; X277A; X286C/H; X312N; X314N;X316A/C/F/N/S/T; X317L; X319N; X323T; X358K; X366H; X383C/F/I/L/M/T/V;X395P; X399A; X414I; X415A/G/H/L/V; X417V; X424A; X426R; X427Y; X434T;and X450S.

In some embodiments, the engineered transaminase polypeptide is capableof converting the substrate compound (2) to the product compound (1)with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10fold, or more activity relative to the activity of the referencepolypeptide of SEQ ID NO: 4. In some embodiments, the engineeredtransaminase polypeptide capable of converting the substrate compound(2) to the product compound (1) with at least 1.2 fold, 1.5 fold, 2fold, 3 fold, 4 fold, 5 fold, 10 fold, or more activity relative to theactivity of the reference polypeptide of SEQ ID NO:4 comprises an aminoacid sequence having one or more residue differences as compared to SEQID NO:4 selected from: X34T, X107G, X113L, X147H, X155V, X233T/V, X323T,X383I/V, and X450S.

In some embodiments, the engineered transaminase polypeptide capable ofconverting the substrate compound (2) to the product compound (1) withat least 1.2 fold the activity relative to SEQ ID NO:4 comprises anamino acid sequence selected from: SEQ ID NO: 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 78, 82, 84, 86, 88, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, and 204.

In some embodiments, the engineered transaminase polypeptides haveincreased stability to temperature and/or solvents used in theconversion reaction as compared to the reference engineered transaminaseof SEQ ID NO: 4. In some embodiments, the engineered transaminasepolypeptide has at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5fold, 10 fold or more stability than the reference polypeptide of SEQ IDNO: 4, as measured by relative activity at 60° C. compared to activityat 55° C. under the same assay conditions. In some embodiments, theengineered transaminase polypeptide having at least 1.2 fold increasedstability as compared to the polypeptide of SEQ ID NO: 4 comprises anamino acid sequence having one or more residue differences as comparedto SEQ ID NO: 2 selected from: X34T, X107G, X113L, X147H, X155V,X233T/V, X323T, X383I/V, and X450S.

In some embodiments, the engineered transaminase polypeptide is capableof converting at least 90% or more, 91% or more, 92% or more, 93% ormore, 94% or more, or 95% or more of compound (2) to compound (1) in 24h or less, at a substrate loading of at least about 20 g/L under theReaction Conditions B, C, or D of Table 2B. In some embodiments, theengineered transaminase polypeptide is capable of converting at least90% or more of compound (2) to compound (1) in 24 h or less at asubstrate loading of at least about 20 g/L at 55° C. In someembodiments, the engineered transaminase polypeptide capable ofconverting at least 90% or more of compound (2) to compound (1) in 24 hor less at a substrate loading of at least about 20 g/L under conditionsat 55° C. comprises an amino acid sequence selected from SEQ ID NO: 8,26, 40, 148, 156, 160, 170, 172, and 180.

In some embodiments, the engineered polypeptide of the presentdisclosure having transaminase activity, e.g., in the conversion of asubstrate compound (2) to product compound (1), has an amino acidsequence comprising a sequence selected from SEQ ID NO: 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, and 204.

In some embodiments, the engineered transaminase having transaminaseactivity comprises an amino acid sequence having at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204, andthe amino acid residue differences as compared to SEQ ID NO:2 present inany one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204, as providedin Tables 2A and 2B.

In addition to the residue positions specified above, any of theengineered transaminase polypeptides disclosed herein can furthercomprise other residue differences relative to SEQ ID NO:2 at otherresidue positions, i.e., residue positions other than X18, X19, X21,X31, X34, X53, X56, X57 X73, X86, X88, X107, X113, X133, X147, X155,X163, X165, X171, X178, X190, X206, X228, X233, X235, X244, X251, X259,X268, X277, X286, X312, X314, X316, X317, X319, X323, X358, X366, X383,X395, X399, X414, X415, X417, X424, X426, X427, X434, and X450. Residuedifferences at these other residue positions provide for additionalvariations in the amino acid sequence without adversely affecting theability of the polypeptide to carry out the transaminase reaction, suchas the conversion of compound (2) to compound (1). Accordingly, in someembodiments, in addition to the amino acid residue differences of anyone of the engineered transaminase polypeptides selected from SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, and 204, the sequence can further comprise1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, or 1-50residue differences at other amino acid residue positions as compared tothe SEQ ID NO: 2. In some embodiments, the number of amino acid residuedifferences as compared to the reference sequence can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 30, 35, 40, 45 or 50 residue positions. The residue differenceat these other positions can include conservative changes ornon-conservative changes. In some embodiments, the residue differencescan comprise conservative substitutions and non-conservativesubstitutions as compared to the wild-type transaminase polypeptide ofV. fluvialis or the engineered transaminase polypeptide of SEQ ID NO: 2.

Amino acid residue differences at other positions relative to thewild-type V. fluvialis or the reference sequence of SEQ ID NO: 2 and theeffect of these differences on enzyme function are described for otherengineered transaminase polypeptides in patent publicationsWO2010081053, US20100209981, and WO2011159910; Yun et al., 2005, ApplEnviron Micriobiol., 71(8):4220-4224); and Cho et al., 2008, BiotechnolBioeng. 99(2):275-84; all of which are incorporated herein by reference.Accordingly, in some embodiments, one or more of the amino aciddifferences as compared to the sequence of SEQ ID NO: 2 can also beintroduced into an engineered transaminase polypeptide of the presentdisclosure at residue positions selected from X4; X6; X12; X18; X30;X44; X56; X81; X82; X85; X95; X112; X122; X127; X130; X157; X164; X166;X167; X174; X181; X208; X228; X253; X256; X272; X285; X286; X293; X297;X302; X311; X312; X316; X317; X319; X320; X321; X332; X385; X407; X408;X409; X415; X418; X431; X434; X438; X444; and X446. In particular, theamino acid residues at the foregoing positions can be selected from thefollowing: X4R/Q/L; X6R/I/N; X12A/G/K; X18A/V/L/I; X30A; X44A; X56V;X81D; X82H; X85A/S/V/T/N/C/G; X95T; X112I; X122E; X127L;X130G/M/A/V/L/I; X157T; X164N/Q/S/T/G/M/A/V/L/I; X166S; X167K/R;X174E/D; X181R; X208I; X228G/T; X253M; X256A; X272A; X285H; X286N/Q/S/T;X293N/Q/S/T; X297A; X302K; X311V; X312D/E; X316K/H/P; X317L/M/Y;X319Q/G/M/N/V; X320A/K; X321L/M/I; X332N/Q/S/T; X385R; X407S; X408A;X409G; X415M/L; X418V/N/Q/S/T; X431D; X434V; X438L; X444V; and X446V.Guidance on the choice of the amino acid residues at these residuepositions and their effect on desirable enzyme properties can be foundin the cited references.

In some embodiments, the present disclosure also provides engineeredtransaminase polypeptides that comprise a fragment of any of theengineered polypeptides described herein that retains the functionalactivity and/or improved property of that engineered transaminase.Accordingly, in some embodiments, the present disclosure provides apolypeptide fragment having transaminase activity, such as in convertingcompound (2) to compound (1) under suitable reaction conditions, whereinthe fragment comprises at least about 80%, 90%, 95%, 96%, 97%, 98%, or99% of a full-length amino acid sequence of an engineered transaminasepolypeptide of the present disclosure, such as an exemplary engineeredtransaminase polypeptide selected from SEQ ID NO: 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, and 204.

In some embodiments, the engineered transaminase polypeptide can have anamino acid sequence comprising a deletion of any one of the engineeredtransaminase polypeptides described herein, such as the exemplaryengineered polypeptides of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and204. Thus, for each and every embodiment of the engineered transaminasepolypeptides of the disclosure, the amino acid sequence can comprisedeletions of one or more amino acids, 2 or more amino acids, 3 or moreamino acids, 4 or more amino acids, 5 or more amino acids, 6 or moreamino acids, 8 or more amino acids, 10 or more amino acids, 15 or moreamino acids, or 20 or more amino acids, up to 10% of the total number ofamino acids, up to 10% of the total number of amino acids, up to 20% ofthe total number of amino acids, or up to 30% of the total number ofamino acids of the transaminase polypeptides, where the associatedfunctional activity and/or improved properties of the engineeredtransaminase described herein is maintained. In some embodiments, thedeletions can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or1-50 amino acid residues. In some embodiments, the number of deletionscan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acidresidues. In some embodiments, the deletions can comprise deletions of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22,23, 24, or 25 amino acid residues.

In some embodiments, the engineered transaminase polypeptide herein canhave an amino acid sequence comprising an insertion as compared to anyone of the engineered transaminase polypeptides described herein, suchas the exemplary engineered polypeptides of SEQ ID NO: 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, and 204. Thus, for each and every embodiment of thetransaminase polypeptides of the disclosure, the insertions can compriseone or more amino acids, 2 or more amino acids, 3 or more amino acids, 4or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 ormore amino acids, 10 or more amino acids, 15 or more amino acids, 20 ormore amino acids, 30 or more amino acids, 40 or more amino acids, or 50or more amino acids, where the associated functional activity and/orimproved properties of the engineered transaminase described herein ismaintained. The insertions can be to amino or carboxy terminus, orinternal portions of the transaminase polypeptide.

In some embodiments, the engineered transaminase polypeptide herein canhave an amino acid sequence comprising a sequence selected from SEQ IDNO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, and 204, and optionally one orseveral (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions,insertions and/or substitutions. In some embodiments, the amino acidsequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or1-50 amino acid residue deletions, insertions and/or substitutions. Insome embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

In some embodiments, the present disclosure provides an engineeredpolypeptide having transaminase activity, which polypeptide comprises anamino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequenceselected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204, with theproviso that the amino acid sequence is not identical to (that is, itexcludes) any of the exemplary engineered transaminase polypeptide aminoacid sequences disclosed in patent application publicationsWO2010081053, US20100209981, and WO2011159910; Yun et al., 2005, ApplEnviron Micriobiol., 71(8):4220-4224); and Cho et al., 2008, BiotechnolBioeng. 99(2):275-84; all of which are incorporated by reference herein.

In the above embodiments, the suitable reaction conditions for theengineered polypeptides can be those described in Tables 2A and 2B, theExamples, and elsewhere herein.

In some embodiments, the polypeptides of the disclosure can be in theform of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purification sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

It is to be understood that the polypeptides described herein are notrestricted to the genetically encoded amino acids. In addition to thegenetically encoded amino acids, the polypeptides described herein maybe comprised, either in whole or in part, of naturally-occurring and/orsynthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the engineered transaminase polypeptides can beprovided on a solid support, such as a membrane, resin, solid carrier,or other solid phase material. A solid support can be composed oforganic polymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A solid support can also be inorganic,such as glass, silica, controlled pore glass (CPG), reverse phase silicaor metal, such as gold or platinum. The configuration of a solid supportcan be in the form of beads, spheres, particles, granules, a gel, amembrane or a surface. Surfaces can be planar, substantially planar, ornon-planar. Solid supports can be porous or non-porous, and can haveswelling or non-swelling characteristics. A solid support can beconfigured in the form of a well, depression, or other container,vessel, feature, or location.

In some embodiments, the engineered polypeptides having transaminaseactivity of the present disclosure can be immobilized on a solid supportsuch that they retain their improved activity, stereoselectivity, and/orother improved properties relative to the reference polypeptide of SEQID NO: 4. In such embodiments, the immobilized polypeptides canfacilitate the biocatalytic conversion of the substrate compounds ofFormula (II) or other suitable substrates, to the product compound ofFormula (I), or corresponding product (e.g., as shown in Schemes 4-8described herein), and after the reaction is complete are easilyretained (e.g., by retaining beads on which polypeptide is immobilized)and then reused or recycled in subsequent reactions. Such immobilizedenzyme processes allow for further efficiency and cost reduction.Accordingly, it is further contemplated that any of the methods of usingthe engineered transaminase polypeptides of the present disclosure canbe carried out using the same engineered transaminase polypeptides boundor immobilized on a solid support.

Methods of enzyme immobilization are well-known in the art. Theengineered transaminase polypeptide can be bound non-covalently orcovalently. Various methods for conjugation and immobilization ofenzymes to solid supports (e.g., resins, membranes, beads, glass, etc.)are well known in the art and described in e.g., Yi et al., “Covalentimmobilization of ω-transaminase from Vibrio fluvialis JS17 on chitosanbeads,” Process Biochemistry 42(5): 895-898 (May 2007); Martin et al.,“Characterization of free and immobilized (5)-aminotransferase foracetophenone production,” Applied Microbiology and Biotechnology 76(4):843-851 (Sep. 2007); Koszelewski et al., “Immobilization ofω-transaminases by encapsulation in a sol-gel/celite matrix,” Journal ofMolecular Catalysis B: Enzymatic, 63: 39-44 (Apr. 2010); Truppo et al.,“Development of an Improved Immobilized CAL-B for the EnzymaticResolution of a Key Intermediate to Odanacatib,” Organic ProcessResearch & Development, published online: dx.doi.org/10.1021/op200157c;Hermanson, G. T., Bioconjugate Techniques, Second Edition, AcademicPress (2008); Mateo et al., “Epoxy sepabeads: a novel epoxy support forstabilization of industrial enzymes via very intense multipoint covalentattachment,” Biotechnology Progress 18(3):629-34 (2002); andBioconjugation Protocols: Strategies and Methods, In Methods inMolecular Biology, C. M. Niemeyer ed., Humana Press (2004); thedisclosures of each which are incorporated by reference herein. Solidsupports useful for immobilizing the engineered transaminases of thepresent disclosure include but are not limited to beads or resinscomprising polymethacrylate with epoxide functional groups,polymethacrylate with amino epoxide functional groups, styrene/DVBcopolymer or polymethacrylate with octadecyl functional groups.Exemplary solid supports useful for immobilizing the engineeredtransaminases of the present disclosure include, but are not limited to,chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi), including thefollowing different types of SEPABEAD: EC-EP, EC-HFA/S, EXA252, EXE119and EXE120.

In some embodiments, the engineered polypeptides can be in variousforms, for example, such as an isolated preparation, as a substantiallypurified enzyme, whole cells transformed with gene(s) encoding theenzyme, and/or as cell extracts and/or lysates of such cells. Theenzymes can be lyophilized, spray-dried, precipitated or be in the formof a crude paste, as further discussed below.

In some embodiments, the polypeptide described herein can be provided inthe form of kits. The enzymes in the kits may be present individually oras a plurality of enzymes. The kits can further include reagents forcarrying out the enzymatic reactions, substrates for assessing theactivity of enzymes, as well as reagents for detecting the products. Thekits can also include reagent dispensers and instructions for use of thekits.

In some embodiments, the polypeptides can be provided on the solidsupport in the form of an array in which the polypeptides are arrangedin positionally distinct locations. The array can be used to test avariety of substrate compounds for conversion by the polypeptides. Aplurality of supports can be configured on an array at variouslocations, addressable for robotic delivery of reagents, or by detectionmethods and/or instruments. Various methods for conjugation tosubstrates, e.g., membranes, beads, glass, etc. are described in, amongothers, Hermanson, G. T., Bioconjugate Techniques, 2^(nd) Edition,Academic Press; (2008), and Bioconjugation Protocols: Strategies andMethods, In Methods in Molecular Biology, C. M. Niemeyer ed., HumanaPress (2004); the disclosures of which are incorporated herein byreference.

In some embodiments, the kits of the present disclosure include arrayscomprising a plurality of different engineered ketoreductasepolypeptides disclosed herein at different addressable position, whereinthe different polypeptides are different variants of a referencesequence each having at least one different improved enzyme property.Such arrays comprising a plurality of engineered polypeptides andmethods of their use are described in WO2009008908.

5.4 Polynucleotides Encoding Engineered Polypeptides, Expression Vectorsand Host Cells

In another aspect, the present disclosure provides polynucleotidesencoding the engineered transaminase polypeptides described herein. Thepolynucleotides may be operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered transaminase can be introduced into appropriate hostcells to express the corresponding transaminase polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the improved transaminase enzymes. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the amino acid sequencespresented in Tables 2A and 2B, and disclosed in the sequence listingincorporated by reference herein as SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, and 204.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used for expression in bacteria; preferredcodons used in yeast are used for expression in yeast; and preferredcodons used in mammals are used for expression in mammalian cells. Insome embodiments, all codons need not be replaced to optimize the codonusage of the transaminases since the natural sequence will comprisepreferred codons and because use of preferred codons may not be requiredfor all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the transaminase enzymes may contain preferredcodons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codonpositions of the full length coding region.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having transaminase activity with the propertiesdisclosed herein, such as the ability to convert substrate compound (2)to product compound (1), where the polypeptide comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a referencesequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204, andone or more residue differences as compared to the reference polypeptideof SEQ ID NO:2 at residue positions selected from X19, X21, X34, X53,X56, X73, X86, X88, X107, X113, X133, X147, X155, X165, X171, X178,X233, X251, X259, X268, X277, X286, X312, X316, X317, X323, X358, X366,X383, X399, X414, X415, X417, X426, X434, and X450, wherein the residuedifferences at residue positions X21, X56, X86, X88, X107, X113, X133,X147, X233, X286, X312, X316, X323, X383, X415, X417, and X434, areselected from: X21H, X56A/C, X86C, X88H/Y, X107G, X113L/P, X133A,X147HN, X233V, X286C/H, X312N, X316C/F/G/N/S/T, X323A, X383C/F/I/M/T,X415A/G/H/L/V, X417V, and X434T. In some embodiments, the specific aminoacid differences at positions X19, X34, X53, X73, X155, X165, X171,X178, X251, X259, X268, X277, X317, X358, X366, X399, X414, X426, andX450 are selected from: X19W, X34A, X53M, X73R, X155V, X165F, X171Q,X178W, X251V, X259V, X268A, X277A, X317L, X358K, X366H, X399A, X414I,X426R, and X450S. In some embodiments, the reference sequence isselected from SEQ ID NO: 4, 8, 26, 36, 40, 78, 100, 102, 148, 156, 160,170, 172, 180, and 198. In some embodiments, the reference sequence isSEQ ID NO: 4. In some embodiments, the reference sequence is SEQ IDNO:100. In some embodiments, the reference sequence is SEQ ID NO:148. Insome embodiments, the reference sequence is SEQ ID NO:156. In someembodiments, the reference sequence is SEQ ID NO:160. In someembodiments, the reference sequence is SEQ ID NO:180.

In some embodiments, the polynucleotide encodes an engineeredpolypeptide having transaminase activity with the properties disclosedherein, wherein the polypeptide comprises an amino acid sequence havingat least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQID NO:2 and one or more residue differences as compared to SEQ ID NO: 2at residue positions selected from X19, X21, X34, X53, X56, X73, X86,X88, X107, X113, X133, X147, X155, X165, X171, X178, X233, X251, X259,X268, X277, X286, X312, X316, X317, X323, X358, X366, X383, X399, X414,X415, X417, X426, X434, and X450, wherein the residue differences atresidue positions X21, X56, X86, X88, X107, X113, X133, X147, X233,X286, X312, X316, X323, X383, X415, X417, and X434, are selected from:X21H, X56A/C, X86C, X88H/Y, X107G, X113L/P, X133A, X147H/V, X233V,X286C/H, X312N, X316C/F/G/N/S/T, X323A, X383C/F/I/M/T, X415A/G/H/L/V,X417V, and X434T.

In some embodiments, the polynucleotide encodes an engineeredpolypeptide having transaminase activity, wherein the polypeptidecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to reference sequence SEQ ID NO:2 and at least thefollowing combination of residue differences as compared to SEQ ID NO:2: X34A, X56A, X57L, X86S, X88A; X153C, X155V, X163F, X315G, and X417T.In some embodiments, the polynucleotide encodes a polypeptide thatfurther comprises combination of residue differences as compared to SEQID NO: 2 selected from: (a) X31M, X57F, X316N, X323T, and X383V; (b)X31M, X57F, X107G, X113L, X233T, X316N, X415H, and X450S; (c) X31M,X57F, X233V, X316N, X323T, X383I, X415H, and X450S; and (d) X31M, X57F,X147H, X316N, X323T, X383I, X415H, and X450S.

In some embodiments, the polynucleotide encodes an engineeredpolypeptide having transaminase activity, wherein the polypeptidecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto a reference polypeptide selected from any one of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, and 204, with the proviso that the amino acidsequence comprises any one of the set of residue differences as comparedto SEQ ID NO: 2 contained in any one of the polypeptide sequences of SEQID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, and 204, as listed in Tables 2A and2B.

In some embodiments, the polynucleotide encoding the engineeredtransaminase comprises a polynucleotide sequence selected from SEQ IDNO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, and 203.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, and 203, or a complementthereof, and encodes a polypeptide having transaminase activity with oneor more of the improved properties described herein. In someembodiments, the polynucleotide capable of hybridizing under highlystringent conditions encodes a transaminase polypeptide comprising anamino acid sequence that has one or more residue differences as comparedto SEQ ID NO: 2 at residue positions selected from: X19, X21, X34, X53,X56, X73, X86, X88, X107, X113, X133, X147, X155, X165, X171, X178,X233, X251, X259, X268, X277, X286, X312, X316, X317, X323, X358, X366,X383, X399, X414, X415, X417, X426, X434, and X450, wherein the residuedifferences at residue positions X21, X56, X86, X88, X107, X113, X133,X147, X233, X286, X312, X316, X323, X383, X415, X417, and X434, areselected from: X21H, X56A/C, X86C, X88H/Y, X107G, X113L/P, X133A,X147H/V, X233V, X286C/H, X312N, X316C/F/G/N/S/T, X323A, X383C/F/I/M/T,X415A/G/H/LN, X417V, and X434T.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered transaminase. In someembodiments, the reference polynucleotide sequence is selected from SEQID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, and 203.

An isolated polynucleotide encoding any of the engineered transaminasepolypeptides herein may be manipulated in a variety of ways to providefor expression of the polypeptide. In some embodiments, thepolynucleotides encoding the polypeptides can be provided as expressionvectors where one or more control sequences is present to regulate theexpression of the polynucleotides and/or polypeptides. Manipulation ofthe isolated polynucleotide prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying polynucleotides and nucleic acid sequencesutilizing recombinant DNA methods are well known in the art. Guidance isprovided in Sambrook et al., 2001, Molecular Cloning: A LaboratoryManual, 3^(rd) Ed., Cold Spring Harbor Laboratory Press; and CurrentProtocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates,1998, updates to 2006.

In some embodiments, the control sequences include among others,promoter, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator.Suitable promoters can be selected based on the host cells used. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus lichenifonnis alpha-amylase gene (amyL), Bacillusstearothennophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. NatlAcad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer etal., 1983, Proc. Natl Acad. Sci. USA 80: 21-25). Exemplary promoters forfilamentous fungal host cells, include promoters obtained from the genesfor Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-likeprotease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of thepromoters from the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), and mutant, truncated,and hybrid promoters thereof. Exemplary yeast cell promoters can be fromthe genes can be from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. For example, exemplary transcription terminatorsfor filamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. Any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used for expression of the engineeredpolypeptides. Effective signal peptide coding regions for bacterial hostcells are the signal peptide coding regions obtained from the genes forBacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57:109-137.Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase. Usefulsignal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is referred to as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila lactase (WO 95/33836). Whereboth signal peptide and propeptide regions are present at the aminoterminus of a polypeptide, the propeptide region is positioned next tothe amino terminus of a polypeptide and the signal peptide region ispositioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

In another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered transaminase polypeptide, and one or more expressionregulating regions such as a promoter and a terminator, a replicationorigin, etc., depending on the type of hosts into which they are to beintroduced. The various nucleic acid and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thepolypeptide at such sites. Alternatively, the nucleic acid sequence ofthe present disclosure may be expressed by inserting the nucleic acidsequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector preferably contains one or more selectablemarkers, which permit easy selection of transformed cells. A selectablemarker is a gene the product of which provides for biocide or viralresistance, resistance to heavy metals, prototrophy to auxotrophs, andthe like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus lichenifonnis, or markers, whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol (Example 1) or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an engineered transaminasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe transaminase enzyme in the host cell. Host cells for use inexpressing the polypeptides encoded by the expression vectors of thepresent invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Vibrio fluvialis,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. An exemplary host cells are Escherichiacoli W3110 (ΔfhuA) and BL21.

Accordingly, in another aspect, the present disclosure provides methodsof manufacturing the engineered transaminase polypeptides, where themethod can comprise culturing a host cell capable of expressing apolynucleotide encoding the engineered transaminase polypeptide underconditions suitable for expression of the polypeptide. The method canfurther comprise isolated or purifying the expressed transaminasespolypeptide, as described herein.

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the transaminase may be introduced into cells byvarious methods known in the art. Techniques include, among others,electroporation, biolistic particle bombardment, liposome mediatedtransfection, calcium chloride transfection, and protoplast fusion.

For the embodiments herein, the engineered polypeptides andcorresponding polynucleotides can be obtained using methods used bythose skilled in the art. The parental polynucleotide sequence encodingthe wild-type polypeptide of Vibrio fluvialis is described in Shin etal., 2003, Appl. Microbiol. Biotechnol. 61(5-6):463-471, and methods ofgenerating engineered transaminase polypeptides with improved stabilityand substrate recognition properties are disclosed in patent applicationpublications WO2010081053 and US20100209981, incorporated herein byreference.

The engineered transaminases with the properties disclosed herein can beobtained by subjecting the polynucleotide encoding the naturallyoccurring or engineered transaminase to mutagenesis and/or directedevolution methods, as discussed above. An exemplary directed evolutiontechnique is mutagenesis and/or DNA shuffling as described in Stemmer,1994, Proc Natl Acad Sci USA 91:10747-10751; WO 95/22625; WO 97/0078; WO97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No.6,537,746. Other directed evolution procedures that can be used include,among others, staggered extension process (StEP), in vitro recombination(Zhao et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR(Caldwell et al., 1994, PCR Methods Appl. 3:S136-S140), and cassettemutagenesis (Black et al., 1996, Proc Natl Acad Sci USA 93:3525-3529).Mutagenesis and directed evolution techniques useful for the purposesherein are also described in the following references: Ling, et al.,1997, Anal. Biochem. 254(2):157-78; Dale et al., 1996,“Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod,” In Methods Mol. Biol. 57:369-74; Smith, 1985, Ann. Rev. Genet.19:423-462; Botstein et al., 1985, Science 229:1193-1201; Carter, 1986,Biochem. J. 237:1-7; Kramer et al., 1984, Cell, 38:879-887; Wells etal., 1985, Gene 34:315-323; Minshull et al., 1999, Curr Opin Chem Biol3:284-290; Christians et al., 1999, Nature Biotech 17:259-264; Crameriet al., 1998, Nature 391:288-291; Crameri et al., 1997, Nature Biotech15:436-438; Zhang et al., 1997, Proc Natl Acad Sci USA 94:45-4-4509;Crameri et al., 1996, Nature Biotech 14:315-319; Stemmer, 1994, Nature370:389-391; Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767and U.S. Pat. No. 6,537,746. All publications are incorporated herein byreference.

The clones obtained following mutagenesis treatment can be screened forengineered transaminases having a desired improved enzyme property. Forexample, where the improved enzyme property desired is thermostability,enzyme activity may be measured after subjecting the enzyme preparationsto a defined temperature and measuring the amount of enzyme activityremaining after heat treatments. Clones containing a polynucleotideencoding a transaminase are then isolated, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using the standard biochemistry techniques, such as HPLCanalysis following derivatization, e.g., with OPA, of the product amine.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides disclosedherein can be prepared by chemical synthesis using, e.g., the classicalphosphoramidite method described by Beaucage et al., 1981, Tet Lett22:1859-69, or the method described by Matthes et al., 1984, EMBO J.3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors.

Accordingly, in some embodiments, a method for preparing the engineeredtransaminase polypeptide can comprise: (a) synthesizing a polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, and 204 and having one or moreresidue differences as compared to SEQ ID NO: 2 at residue positionsselected from: X19, X21, X34, X53, X56, X73, X86, X88, X107, X113, X133,X147, X155, X165, X171, X178, X233, X251, X259, X268, X277, X286, X312,X316, X317, X323, X358, X366, X383, X399, X414, X415, X417, X426, X434,and X450, wherein the residue differences at residue positions X21, X56,X86, X88, X107, X113, X133, X147, X233, X286, X312, X316, X323, X383,X415, X417, and X434, are selected from: X21H, X56A/C, X86C, X88H/Y,X107G, X113L/P, X133A, X147HN, X233V, X286C/H, X312N, X316C/F/G/N/S/T,X323A, X383C/F/I/M/T, X415A/G/H/L/V, X417V, and X434T; and (b)expressing the transaminase polypeptide encoded by the polynucleotide.In some embodiments of the method, the residue differences at residuepositions X19, X34, X53, X73, X155, X165, X171, X178, X251, X259, X268,X277, X317, X358, X366, X399, X414, X426, and X450 are selected fromX19W, X34A, X53M, X73R, X155V, X165F, X171Q, X178W, X251V, X259V, X268A,X277A, X317L, X358K, X366H, X399A, X414I, X426R, and X450S.

In some embodiments of the method, the amino acid sequence encoded bythe polynucleotide can optionally have one or several (e.g., up to 3, 4,5, or up to 10) amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35,40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

The expressed engineered transaminase can be measured for the desiredimproved property, e.g., activity, enantioselectivity, stability, andproduct tolerance, in the conversion of compound (2) to compound (1) byany of the assay conditions described herein.

In some embodiments, any of the engineered transaminase enzymesexpressed in a host cell can be recovered from the cells and or theculture medium using any one or more of the well known techniques forprotein purification, including, among others, lysozyme treatment,sonication, filtration, salting-out, ultra-centrifugation, andchromatography. Suitable solutions for lysing and the high efficiencyextraction of proteins from bacteria, such as E. coli, are provided inTable 2A and the Examples, and also commercially available, e.g.,CelLytic B™ from Sigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the transaminase polypeptideinclude, among others, reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, gel electrophoresis,and affinity chromatography. Conditions for purifying a particularenzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved transaminase enzymes. For affinity chromatography purification,any antibody which specifically binds the transaminase polypeptide maybe used. For the production of antibodies, various host animals,including but not limited to rabbits, mice, rats, etc., may be immunizedby injection with a transaminase polypeptide, or a fragment thereof. Thetransaminase polypeptide or fragment may be attached to a suitablecarrier, such as BSA, by means of a side chain functional group orlinkers attached to a side chain functional group.

5.7 Methods of Using the Engineered Transaminase Enzymes

As noted above, the engineered transaminase polypeptides of the presentdisclosure were evolved to efficiently convert the ketone of theexemplary substrate compound (2) to the corresponding chiral amine ofthe exemplary product compound (1) in diastereomeric excess, in thepresence of an amino donor under suitable reaction conditions. Thestructural features of the engineered transaminase polypeptides alsoallow for the conversion of large prochiral ketone substrate compounds,other than compound (2), to their corresponding chiral amine compoundsin stereomeric excess. Accordingly, in another aspect, the presentdisclosure provides processes using the engineered transaminasepolypeptides to carry out a transamination reaction in which an aminogroup from an amino donor is transferred to an amino acceptor, e.g., aketone substrate compound, to produce an amine compound. Generally, theprocess for performing the transamination reaction comprises contactingor incubating an engineered transaminase polypeptide of the disclosurewith an amino acceptor (e.g., a ketone substrate compound) and an aminodonor (e.g., isopropylamine) with under reaction conditions suitable forconverting the amino acceptor to an amine compound.

In some embodiments, the present disclosure provides a process for thepreparation of an amine compound of Formula (I)

wherein

Ring A is a 6-membered carbocyclic ring, optionally including anunsaturated C—C bond between positions 2 and 3 and/or positions 5 and 6,and/or optionally substituted independently positions 2, 3, 4, 5 and 6with a group selected from halo, hydroxy, and methyl;

Ring B is a 6-membered carbocyclic ring, optionally including anunsaturated C—C bond between positions 5 and 10, and/or optionallysubstituted independently at one or more of positions 9 and 10 with agroup selected from halo, hydroxy, and methyl;

Ring C is a 5- or 6-membered carbocyclic ring (i.e., m=0 or 1),optionally substituted at position 10 with a group selected from halo,hydroxy, methyl, ethyl, and carbonyl;

Ring D is a 5-, 6-, or 7-membered carbocyclic ring (i.e., n=0, 1, or 2),optionally including 1, 2, or 3 unsaturated C—C bonds, and/or optionallysubstituted independently as follows:

at position 14 with a group selected from halo, hydroxy, amino, carboxy,cyano, nitro, thio, straight-chain or branched (C₁-C₄)alkyl,straight-chain or branched (C₁-C₄)alkenyl, straight-chain or branched(C₁-C₃)alkylamino, and cyclopropyl bridging to position 12;

at position 15 or position 16 with a group selected from halo, hydroxy,amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionallysubstituted (C₁-C₆)alkylamino, optionally substituted(C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionallysubstituted (C₁-C₆)alkylsulfonyl, optionally substituted(C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl,(C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl,aminocarbonyl(C₁-C₆)alkyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryloxy, optionallysubstituted acylamino, optionally substituted arylthio, optionallysubstituted arylsulfonyl, optionally substituted arylsulfinyl,optionally substituted aryloxycarbonyl, optionally substitutedarylcarbonyloxy, optionally substituted heteroaryloxy, optionallysubstituted heteroarylamino, optionally substituted heteroarylthio,optionally substituted heteroarylsulfonyl, optionally substitutedheteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl,optionally substituted heteroarylcarbonyloxy,alkylaminosulfonyl(C₁-C₆)alkyl, arylsulfonyl(C₁-C₆)alkyl, andheteroarylsulfonyl(C₁-C₆)alkyl;

with the proviso that the compound of Formula (I) is not compound (1)

wherein the method comprises contacting the ketone substrate compound ofFormula (II),

wherein rings A, B, C, and D are as defined above for the compound ofFormula (I),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting cyclopamine analog compounds ofFormula (IIa), wherein Ring C is a 5-membered carbocyclic ring,optionally substituted at position 11, and Ring D is a 7-memberedcarbocyclic ring substituted at position 16, which can be converted toan amine product compound of Formula (Ia) as in Scheme 5.

Accordingly, in some embodiments, the present disclosure provides aprocess for preparation of an amine compound of Formula (Ia)

wherein

Rings A and B comprise one of the following:

-   -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D comprises an unsaturated C—C bond between positions 12 and 14;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl;

with the proviso that the compound of Formula (I) is not compound (1)

-   -   wherein the method comprises contacting the ketone substrate        compound of Formula (IIa),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined abovefor the compound of Formula (Ia),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting cyclopamine analog compounds ofFormula (IIb), wherein Ring C is 5-membered carbocyclic ring and Ring Dis a 6-membered carbocyclic ring, to the chiral amine product compoundof Formula (Ib) as shown in Scheme 6:

Accordingly, in some embodiments, the present disclosure provides aprocess for preparation of an amine compound of Formula (Ib)

wherein

-   -   Rings A and B comprise one of the following:    -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D comprises an unsaturated C—C bond between positions 12 and 14, ora bridging cyclopropyl between positions 12 and 14;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl;

wherein the method comprises contacting the ketone substrate compound ofFormula (IIb),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined abovefor the compound of Formula (Ib),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments, the engineered transaminase polypeptides can beused to prepare any of the cyclopamine analog compounds disclosed in WO2011017551A1, published Feb. 10, 2011, which is hereby incorporated byreference herein.

Numerous other cyclopamine analog compounds (other than thoseencompassed by Formulas (Ia) and (Ib)) are known in the art. In someembodiments, it is contemplated that the engineered transaminasepolypeptides of the present disclosure can be used in biocatalyticprocesses to prepare any of the known veratramine analog compounds.

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting veratramine analog compounds ofFormula (IIc), wherein Ring C is 5-membered carbocyclic ring and Ring Dis a 6-membered carbocyclic ring, to the chiral amine product compoundof Formula (Ic) as shown in Scheme 7:

Accordingly, in some embodiments, the present disclosure provides aprocess for preparation of an amine compound of Formula (Ic)

wherein

-   -   Rings A and B comprise one of the following:    -   (a) an unsaturated C—C bond between positions 5 and 6;    -   (b) an unsaturated C—C bond between positions 5 and 10;    -   (c) a hydrogen at position 5 cis to the methyl group at position        4; or    -   (d) a hydrogen at position 5 trans to the methyl group at        position 4;

Ring D is aromatic;

R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, andcarbonyl;

R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain orbranched (C₁-C₄)alkenyl, and straight-chain or branched(C₁-C₃)alkylamino; and

R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano,nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,optionally substituted(C₁-C₆)alkyloxy, optionally substituted(C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino,optionally substituted (C₁-C₆)alkylthio, optionally substituted(C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy,optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl;

wherein the method comprises contacting the ketone substrate compound ofFormula (IIc),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined abovefor the compound of Formula (Ic),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

Numerous other veratramine analog compounds (other than thoseencompassed by Formula (Ic)) are known in the art. In some embodiments,it is contemplated that the engineered transaminase polypeptides of thepresent disclosure can be used in biocatalytic processes to prepare anyof the known veratramine analog compounds.

In some embodiments, the engineered transaminase polypeptides of thedisclosure are capable of converting steroid analog compounds of Formula(IId), wherein Ring C is 6-membered carbocyclic ring and Ring D is a5-membered carbocyclic ring, to a chiral amine product of Formula (Id)as shown in Scheme 8:

Accordingly, in some embodiments, the present disclosure provides aprocess for preparation of an amine compound of Formula (Id)

wherein

Ring A comprises an unsaturated C—C bond between positions 2 and 3, orpositions 5 and 6;

R¹ and R² are selected independently from hydrogen, halo, hydroxy,amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionallysubstituted (C₁-C₆)alkylamino, optionally substituted(C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionallysubstituted (C₁-C₆)alkylsulfonyl, optionally substituted(C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl,(C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl,aminocarbonyl(C₁-C₆)alkyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aryloxy, optionallysubstituted arylamino, optionally substituted arylthio, optionallysubstituted arylsulfonyl, optionally substituted arylsulfinyl,optionally substituted aryloxycarbonyl, optionally substitutedarylcarbonyloxy, optionally substituted heteroaryloxy, optionallysubstituted heteroarylamino, optionally substituted heteroarylthio,optionally substituted heteroarylsulfonyl, optionally substitutedheteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl,optionally substituted heteroarylcarbonyloxy,alkylaminosulfonyl(C₁-C₆)alkyl, arylsulfonyl(C₁-C₆)alkyl, andheteroarylsulfonyl(C₁-C₆)alkyl;

R³, R⁴, and R⁵ are selected independently from hydrogen, halo, hydroxy,amino, carboxy, cyano, nitro, thio, straight-chain or branched(C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, andstraight-chain or branched (C₁-C₃)alkylamino; and

R⁶, R⁷, and R⁸ are selected independently from hydrogen, halo, hydroxy,and methyl;

wherein the method comprises contacting the ketone substrate compound ofFormula (IId),

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are as defined above for thecompound of Formula (Id),

with an engineered transaminase polypeptide of the present disclosure inthe presence of an amino donor under suitable reaction conditions.

In some embodiments of the process for preparing an amine compound ofFormula (Id), the process can be carried out using a ketone substratecompound of Formula (IId) selected from those shown in Table 3.

TABLE 3 Ketone substrate compound of Formula (iid) Chiral amine productcompound of Formula (Id)

In addition to the compounds of Formula (IId), including those shown inTable 3, there are a multitude of steroid analog compounds known in theart. The present disclosure contemplates that any steroid analogcompound with a ketone group at position 1 of Ring A could be used asketone substrates with an engineered transaminase polypeptides of thepresent disclosure in a process to prepare its corresponding steroidanalog compound with a chiral amine group at position 1.

In view of the stereoselectivity of the engineered transaminasepolypeptides of the present disclosure, in some embodiments the processresults in the formation of the chiral amine compounds of Formula (I),Formula (Ia), Formula (Ib), Formula (Ic), and Formula (Id) indiastereomeric excess. In some embodiments, the process results in theformation of the chiral amine compound of Formula (I), Formula (Ia),Formula (Ib), Formula (Ic), and Formula (Id) in diastereomeric excess ofat least 90%, 95%, 96%, 97%, 98%, 99%, or greater.

For the foregoing processes, any of the engineered transaminasepolypeptides described herein can be used. By way of example and withoutlimitation, in some embodiments, the process can use an engineeredpolypeptide having transaminase activity of the present disclosurecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity to a reference sequence selected from SEQ ID NO: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, and 204, and one or more residue differences as comparedto SEQ ID NO:2 at residue positions selected from X19, X21, X34, X53,X56, X73, X86, X88, X107, X113, X133, X147, X155, X165, X171, X178,X233, X251, X259, X268, X277, X286, X312, X316, X317, X323, X358, X366,X383, X399, X414, X415, X417, X426, X434, and X450, wherein the residuedifferences at residue positions X21, X56, X86, X88, X107, X113, X133,X147, X233, X286, X312, X316, X323, X383, X415, X417, and X434, areselected from: X21H, X56A/C, X86C, X88H/Y, X107G, X113L/P, X133A,X147H/V, X233V, X286C/H, X312N, X316C/F/G/N/S/T, X323A, X383C/F/I/M/T,X415A/G/H/L/V, X417V, and X434T. In some embodiments, the specific aminoacid differences at positions X19, X34, X53, X73, X155, X165, X171,X178, X251, X259, X268, X277, X317, X358, X366, X399, X414, X426, andX450 are selected from: X19W, X34A, X53M, X73R, X155V, X165F, X171Q,X178W, X251V, X259V, X268A, X277A, X317L, X358K, X366H, X399A, X414I,X426R, and X450S. In some embodiments, the reference sequence isselected from SEQ ID NO: 4, 8, 26, 36, 40, 78, 100, 102, 148, 156, 160,170, 172, 180, and 198. In some embodiments, the reference sequence isSEQ ID NO:4. In some embodiments, the reference sequence is SEQ IDNO:100. In some embodiments, the reference sequence is SEQ ID NO:148. Insome embodiments, the reference sequence is SEQ ID NO:156. In someembodiments, the reference sequence is SEQ ID NO:160. In someembodiments, the reference sequence is SEQ ID NO:180.

In some embodiments, exemplary transaminase polypeptides capable ofcarrying out the processes herein can be a polypeptide comprising anamino acid sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, and 204. Guidance on the choice and use of the engineeredtransaminase polypeptides is provided in the descriptions herein, forexample Tables 2A and 2B and the Examples.

In the embodiments herein and illustrated in the Examples, variousranges of suitable reaction conditions that can be used, including butnot limited, to ranges of amino donor, pH, temperature, buffer, solventsystem, substrate loading, polypeptide loading, cofactor loading,pressure, and reaction time. Further suitable reaction conditions forcarrying out the process for biocatalytic conversion of substratecompounds to product compounds using an engineered transaminasepolypeptide described herein can be readily optimized in view of theguidance provided herein by routine experimentation that includes, butis not limited to, contacting the engineered transaminase polypeptideand substrate compound under experimental reaction conditions ofconcentration, pH, temperature, solvent conditions, and detecting theproduct compound.

In some embodiments herein, the transaminase polypeptide uses an aminodonor to form the product compounds. In some embodiments, the aminodonor in the reaction condition can be selected from isopropylamine(also referred to herein as “IPM”), putrescine, L-lysine,α-phenethylamine, D-alanine, L-alanine, or D,L-alanine, orD,L-ornithine. In some embodiments, the amino donor is selected fromIPM, putrescine, L-lysine, D- or L-alanine. In some embodiments, theamino donor is IPM. In some embodiments, the suitable reactionconditions comprise the amino donor, in particular IPM, present at aconcentration of at least about 0.1 to about 3 M, 0.2 to about 2.5 M,about 0.5 to about 2 M or about 1 to about 2 M. In some embodiments, theamino donor is present at a concentration of about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 1, 1.5, 2, 2.5 or 3 M. Higher concentrations ofamino donor, e.g., IPM, can be used to shift the equilibrium towardsamine product formation.

Suitable reaction conditions using the engineered transaminasepolypeptides also typically comprise a cofactor. Cofactors useful fortransaminase enzymes herein include, but are not limited to,pyridoxal-5′-phosphate (also known as pyridoxal-phosphate, PLP, P5P),pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and theirphosphorylated counterparts pyridoxine phosphate (PNP) and pyridoxaminephosphate (PMP). In some embodiments, the cofactor PLP is presentnaturally in the cell extract and does not need to be supplemented. Insome embodiments of the processes, the suitable reaction conditionscomprise exogenous cofactor added to the enzyme reaction mixture, forexample, when using partially purified or purified transaminase enzyme.In some embodiments, the suitable reaction conditions can comprise thepresence of a cofactor selected from PLP, PN, PL, PM, PNP, and PMP, at aconcentration of about 0.1 g/L to about 10 g/L, about 0.2 g/L to about 5g/L, about 0.5 g/L to about 2.5 g/L. In some embodiments, the reactionconditions comprise a PLP concentration of about 0.1 g/L or less, 0.2g/L or less, 0.5 g/L or less, 1 g/L or less, 2.5 g/L or less, 5 g/L orless, or 10 g/L or less. In some embodiments, the cofactor can be addedeither at the beginning of the reaction and/or additional cofactor isadded during the reaction.

Substrate compound in the reaction mixtures can be varied, taking intoconsideration, for example, the desired amount of product compound, theeffect of substrate concentration on enzyme activity, stability ofenzyme under reaction conditions, and the percent conversion ofsubstrate to product. In some embodiments, the suitable reactionconditions comprise a substrate compound loading of at least about 0.5to about 200 g/L, 1 to about 200 g/L, about 5 to about 150 g/L, about 10to about 100 g/L, about 20 to about 100 g/L, or about 50 to about 100g/L. In some embodiments, the suitable reaction conditions comprise asubstrate compound loading of at least about 0.5 g/L, at least about 1g/L, at least about 5 g/L, at least about 10 g/L, at least about 15 g/L,at least about 20 g/L, at least about 30 g/L, at least about 50 g/L, atleast about 75 g/L, at least about 100 g/L, at least about 150 g/L or atleast about 200 g/L, or even greater. The values for substrate loadingsprovided herein are based on the molecular weight of compound (2),however it also contemplated that the equivalent molar amounts ofvarious hydrates and salts of compound (2) also can be used in theprocess. In addition, ketone substrate compounds of Formula (II),including compounds of Formula (IIa), (IIb), (IIc), and (IId) can alsobe used in appropriate amounts, in light of the amounts used forcompound (2).

In carrying out the reactions described herein, the engineeredtransaminase polypeptide may be added to the reaction mixture in theform of a purified enzyme, whole cells transformed with gene(s) encodingthe enzyme, and/or as cell extracts and/or lysates of such cells. Wholecells transformed with gene(s) encoding the engineered transaminaseenzyme or cell extracts, lysates thereof, and isolated enzymes may beemployed in a variety of different forms, including solid (e.g.,lyophilized, spray-dried, and the like) or semisolid (e.g., a crudepaste). The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like), followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde, or immobilization to asolid phase (e.g., Eupergit C, and the like).

The gene(s) encoding the engineered transaminase polypeptides can betransformed into host cell separately or together into the same hostcell. For example, in some embodiments one set of host cells can betransformed with gene(s) encoding one engineered transaminasepolypeptide and another set can be transformed with gene(s) encodinganother engineered transaminase polypeptide. Both sets of transformedcells can be utilized together in the reaction mixture in the form ofwhole cells, or in the form of lysates or extracts derived therefrom. Inother embodiments, a host cell can be transformed with gene(s) encodingmultiple engineered transaminase polypeptide. In some embodiments theengineered polypeptides can be expressed in the form of secretedpolypeptides and the culture medium containing the secreted polypeptidescan be used for the transaminase reaction.

The enhancements in activity and/or stereoselectivity of the engineeredtransaminase polypeptides disclosed herein provide for processes whereinhigher percentage conversion can be achieved with lower concentrationsof the engineered polypeptide. In some embodiments of the process, thesuitable reaction conditions comprise an engineered polypeptideconcentration of about 0.01 to about 50 g/L; about 0.05 to about 50 g/L;about 0.1 to about 40 g/L; about 1 to about 40 g/L; about 2 to about 40g/L; about 5 to about 40 g/L; about 5 to about 30 g/L; about 0.1 toabout 10 g/L; about 0.5 to about 10 g/L; about 1 to about 10 g/L; about0.1 to about 5 g/L; about 0.5 to about 5 g/L; or about 0.1 to about 2g/L. In some embodiments, the transaminase polypeptide is concentrationat about 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40,or 50 g/L.

During the course of the transamination reactions, the pH of thereaction mixture may change. The pH of the reaction mixture may bemaintained at a desired pH or within a desired pH range. This may bedone by adding an acid or base, before and/or during the course of thereaction. Alternatively, the pH may be controlled by using a buffer.Accordingly, in some embodiments, the reaction condition comprises abuffer. Suitable buffers to maintain desired pH ranges are known in theart and include, by way of example and not limitation, borate,carbonate, phosphate, triethanolamine (TEA), and the like. In someembodiments, the buffer is borate. In some embodiments of the process,the suitable reaction conditions comprise a buffer solution of TEA,where the TEA concentration is from about 0.01 to about 0.4 M, 0.05 toabout 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In someembodiments, the reaction condition comprises a TEA concentration ofabout 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12, 0.14, 0.16, 0.18,0.2, 0.3, or 0.4 M. In some embodiments, the reaction conditionscomprise water as a suitable solvent with no buffer present.

In the embodiments of the process, the reaction conditions can comprisea suitable pH. The desired pH or desired pH range can be maintained byuse of an acid or base, an appropriate buffer, or a combination ofbuffering and acid or base addition. The pH of the reaction mixture canbe controlled before and/or during the course of the reaction. In someembodiments, the suitable reaction conditions comprise a solution pHfrom about 6 to about 12, pH from about 6 to about 10, pH from about 6to about 8, pH from about 7 to about 10, pH from about 7 to about 9, orpH from about 7 to about 8. In some embodiments, the reaction conditionscomprise a solution pH of about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 11.5 or 12.

In the embodiments of the processes herein, a suitable temperature canbe used for the reaction conditions, for example, taking intoconsideration the increased reaction rate at higher temperatures, andthe activity of the enzyme during the reaction time period. For example,the engineered polypeptides of the present disclosure have increasedstability relative to naturally occurring transaminase polypeptide e.g.,the wild-type polypeptide of SEQ ID NO: 2, which allow the engineeredpolypeptides to be used at higher temperatures for increased conversionrates and improved substrate solubility characteristics. Accordingly, insome embodiments, the suitable reaction conditions comprise atemperature of about 10° C. to about 70° C., about 10° C. to about 65°C., about 15° C. to about 60° C., about 20° C. to about 60° C., about20° C. to about 55° C., about 30° C. to about 55° C., or about 40° C. toabout 50° C. In some embodiments, the suitable reaction conditionscomprise a temperature of about 10° C., 15° C., 20° C., 25° C., 30° C.,35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C. Insome embodiments, the temperature during the enzymatic reaction can bemaintained at a temperature throughout the course of the reaction oradjusted over a temperature profile during the course of the reaction.

The processes herein are generally carried out in a solvent. Suitablesolvents include water, aqueous buffer solutions, organic solvents,polymeric solvents, and/or co-solvent systems, which generally compriseaqueous solvents, organic solvents and/or polymeric solvents. Theaqueous solvent (water or aqueous co-solvent system) may be pH-bufferedor unbuffered. In some embodiments, the processes are generally carriedout in an aqueous co-solvent system comprising an organic solvent (e.g.,ethanol, isopropanol (IPA), dimethyl sulfoxide (DMSO), ethyl acetate,butyl acetate, 1-octanol, heptane, octane, methyl t-butyl ether (MTBE),toluene, and the like), ionic or polar solvents (e.g., 1 ethyl 4methylimidazolium tetrafluoroborate, 1 butyl 3 methylimidazoliumtetrafluoroborate, 1 butyl 3 methylimidazolium hexafluorophosphate,glycerol, polyethylene glycol, and the like). In some embodiments, theco-solvent can be a polar solvent, such as a polyol, dimethylsulfoxide,DMSO, or lower alcohol. The non-aqueous co-solvent component of anaqueous co-solvent system may be miscible with the aqueous component,providing a single liquid phase, or may be partly miscible or immisciblewith the aqueous component, providing two liquid phases. Exemplaryaqueous co-solvent systems can comprise water and one or moreco-solvents selected from an organic solvent, polar solvent, and polyolsolvent. In general, the co-solvent component of an aqueous co-solventsystem is chosen such that it does not adversely inactivate thetransaminase enzyme under the reaction conditions. Appropriateco-solvent systems can be readily identified by measuring the enzymaticactivity of the specified engineered transaminase enzyme with a definedsubstrate of interest in the candidate solvent system, utilizing anenzyme activity assay, such as those described herein.

In some embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent, where the co-solvent comprises DMSO atabout 1% to about 80% (v/v), about 1 to about 70% (v/v), about 2% toabout 60% (v/v), about 5% to about 40% (v/v), 10% to about 40% (v/v),10% to about 30% (v/v), or about 10% to about 20% (v/v). In someembodiments of the process, the suitable reaction conditions comprise anaqueous co-solvent comprising DMSO at least about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% (v/v). Insome embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent comprising DMSO of from about 15% (v/v)to about 45% (v/v), from about 20% (v/v) to about 30% (v/v), and in someembodiments a DMSO concentration of about 25% (v/v).

In some embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent, where the co-solvent can comprise apolymeric polyol solvent. Examples of suitable polyol solvents include,by way of example and not limitation, polyethylene glycol, polyethyleneglycol methyl ether, diethylene glycol dimethyl ether, triethyleneglycol dimethyl ether, and polypropylene glycol. In some embodiments,the aqueous co-solvent comprises polyethylene glycol, which is availablein different molecular weights. Particularly useful are lower molecularweight polyethylene glycols, such as PEG200 to PEG600. Accordingly, insome embodiments, the aqueous co-solvent can comprise PEG200 of about 1%to about 40% v/v; about 1% to about 40% v/v; about 2% to about 40% v/v;about 5% to about 40% v/v; 2% to about 30% v/v; 5% to about 30% v/v; 1to about 20% v/v; about 2% to about 20% v/v; about 5% to about 20% v/v;about 1% to about 10% v/v; about 2% to about 10% v/v. In someembodiments, the suitable reaction conditions comprises an aqueousco-solvent comprising PEG200 at about 1%, 2%, 5%, 10%, 15%, 20%; 25%;30%; 35%; 35% or about 40% v/v.

The quantities of reactants used in the transamination reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of transaminase substrate employed. Thosehaving ordinary skill in the art will readily understand how to varythese quantities to tailor them to the desired level of productivity andscale of production.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the cofactor,transaminase, and transaminase substrate may be added first to thesolvent.

The solid reactants (e.g., enzyme, salts, substrate compounds, etc.) maybe provided to the reaction in a variety of different forms, includingpowder (e.g., lyophilized, spray dried, and the like), solution,emulsion, suspension, and the like. The reactants can be readilylyophilized or spray dried using methods and equipment that are known tothose having ordinary skill in the art. For example, the proteinsolution can be frozen at −80° C. in small aliquots, then added to apre-chilled lyophilization chamber, followed by the application of avacuum.

For improved mixing efficiency when an aqueous co-solvent system isused, the transaminase and cofactor may be added and mixed into theaqueous phase first. The organic phase may then be added and mixed in,followed by addition of the transaminase substrate. Alternatively, thetransaminase substrate may be premixed in the organic phase, prior toaddition to the aqueous phase.

The transamination reaction is generally allowed to proceed untilfurther conversion of ketone substrate to amine product does not changesignificantly with reaction time, e.g., less than 10% of substrate beingconverted, or less than 5% of substrate being converted. In someembodiments, the reaction is allowed to proceed until there is completeor near complete conversion of substrate ketone to product amine.Transformation of substrate to product can be monitored using knownmethods by detecting substrate and/or product. Suitable methods includegas chromatography, HPLC, and the like. Conversion yields of the chiralamine product generated in the reaction mixture are generally greaterthan about 50%, may also be greater than about 60%, may also be greaterthan about 70%, may also be greater than about 80%, may also be greaterthan 90%, and may be greater than about 97%. In some embodiments, themethods for preparing compounds of Formula (I) using an engineeredtransaminase polypeptide under suitable reaction conditions results inat least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterconversion of ketone substrate, e.g, compound of Formula (II), to theamine product compound, e.g., compound of Formula (I) in about 48 h orless, in about 36 h or less, in about 24 h or less, or even less time.

In some embodiments of the process, the suitable reaction conditionscomprise a substrate loading of at least about 20 g/L, 30 g/L, 40 g/L,50 g/L, 60 g/L, 70 g/L, 100 g/L, or more, and wherein the processresults in at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater conversion of substrate compound to product compound in about 48h or less, in about 36 h or less, or in about 24 h or less.

The engineered transaminase polypeptides of the present disclosure whenused in the process for preparing chiral amine compounds of Formula (I)under suitable reaction conditions result in an diastereomeric excess ofthe chiral amine in at least 90%, 91%, 92%, 93%, 94%, 95% 97%, 98, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% d.e.

In a further embodiment of the processes, the suitable reactionconditions can comprise an initial substrate loading to the reactionsolution which is then contacted by the polypeptide. This reactionsolution is then further supplemented with additional substrate compoundas a continuous addition over time at a rate of at least about 1 g/L/h,at least about 2 g/L/h, at least about 4 g/L/h, at least about 6 g/L/h,or higher. Thus, according to these suitable reaction conditions,polypeptide is added to a solution having an initial substrate loadingof at least about 20 g/L, 30 g/L, or 40 g/L. This addition ofpolypeptide is then followed by continuous addition of further substrateto the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6 g/L/h until amuch higher final substrate loading of at least about 30 g/L, 40 g/L, 50g/L, 60 g/L, 70 g/L, 100 g/L, 150 g/L, 200 g/L or more, is reached.Accordingly, in some embodiments of the process, the suitable reactionconditions comprise addition of the polypeptide to a solution having aninitial substrate loading of at least about 20 g/L, 30 g/L, or 40 g/Lfollowed by addition of further substrate to the solution at a rate ofabout 2 g/L/h, 4 g/L/h, or 6 g/L/h until a final substrate loading of atleast about 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L or more, isreached. This substrate supplementation reaction condition allows forhigher substrate loadings to be achieved while maintaining high rates ofconversion of ketone substrate to amine product of at least about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater. In some embodimentsof this process, the further substrate added is in a solution comprisingisopropylamine or isopropylamine acetate at a concentration of at leastabout 0.5 M, at least about 1.0 M, at least about 2.5 M, at least about5.0 M, at least about 7.5 M, at least about 10.0 M.

In some embodiments of the processes, the transamination reaction cancomprise the following suitable reaction conditions(a) substrate loadingat about 5 g/L to 200 g/L; (b) about 0.1 to 50 g/L of engineeredtransaminase polypeptide; (c) about 0.1 to 4 M of isopropylamine (IPM);(d) about 0.1 to 10 g/L of pyridoxal phosphate (PLP) cofactor; (e) pH ofabout 6 to 9; and (f) temperature of about 30 to 60° C.

In some embodiments of the processes, the transamination reaction cancomprise the following suitable reaction conditions: (a) substrateloading at about 10 g/L to 150 g/L; (b) about 0.5 to 20 g/L ofengineered transaminase polypeptide; (c) about 0.1 to 3 M ofisopropylamine (IPM); (d) about 0.1 to 10 g/L of pyridoxal phosphate(PLP) cofactor; (e) about 0.05 to 0.20 M TEA buffer; (f) about 1% toabout 45% DMSO; (g) pH of about 6 to 9; and (h) temperature of about 30to 65° C.

In some embodiments of the processes, the transamination reaction cancomprise the following suitable reaction conditions: (a) substrateloading at about 20 to 100 g/L; (b) about 1 to 5 g/L of engineeredtransaminase polypeptide; (c) about 0.5 to 2 M of isopropylamine (IPM);(d) about 0.2 to 2 g/L of pyridoxal phosphate (PLP) cofactor; (e) about0.1 M TEA buffer; (f) about 25% DMSO; (e) pH of about 8; and (f)temperature of about 45 to 60° C.

In some embodiments, additional reaction components or additionaltechniques carried out to supplement the reaction conditions. These caninclude taking measures to stabilize or prevent inactivation of theenzyme, reduce product inhibition, and/or shift reaction equilibrium toproduct amine formation.

Accordingly, in some embodiments of the process for preparing an amine,such as a chiral amine, additional quantities of the amino acceptor canbe added (up to saturation) and/or the amino acceptor (ketone) formedcan be continuously removed from the reaction mixture. For example, asolvent bridge or a two phase co-solvent system can be used to move theamine product to an extraction solution, and thereby reduce inhibitionby amine product and also shift the equilibrium towards productformation (see, e.g., Yun and Kim, 2008, Biosci. Biotechnol. Biochem.72(11):3030-3033).

In some embodiments of the processes, the suitable reaction conditionscomprise the presence of the reduced cofactor, nicotinamide adeninedinucleotide (NADH), which can act to limit the inactivation of thetransaminase enzyme (see e.g., van Ophem et al., 1998, Biochemistry37(9):2879-88). In such embodiments where NADH is present, a cofactorregeneration system, such as glucose dehydrogenase (GDH) and glucose orformate dehydrogenase and formate can be used to regenerate the NADH inthe reaction medium.

In some embodiments, the process can further comprise removal of thecarbonyl by-product formed from the amino group donor when the aminogroup is transferred to the amino group acceptor. Such removal in situcan reduce the rate of the reverse reaction such that the forwardreaction dominates and more substrate is then converted to product.Removal of the carbonyl by-product can be done in a number of ways.Where the amino group donor is an amino acid, such as alanine, thecarbonyl by-product, a keto acid, can be removed by reaction with aperoxide (see, e.g., US 2008/0213845, incorporated herein by reference).Peroxides that can be used include, among others, hydrogen peroxide;peroxyacids (peracids), such as peracetic acid (CH₃CO₃H),trifluoroperacetic acid and metachloroperoxybenzoic acid; organicperoxides such as t-butyl peroxide ((CH₃)₃COOH); or other selectiveoxidants such as tetrapropylammonium perruthenate, MnO₂, KMnO₄,ruthenium tetroxide and related compounds. Alternatively, pyruvateremoval can be achieved via its reduction to lactate by employinglactate dehydrogenase to shift equilibrium to the product amine (see,e.g., Koszelewski et al., 2008, Adv. Syn. Catal. 350:2761-2766).Pyruvate removal can also be achieved via its decarboxylation byemploying pyruvate decarboxylase (see, e.g., Höhne et al., 2008, ChemBioChem 9:363-365) or acetolactate synthase (see, e.g., Yun and Kim,supra).

Alternatively, in embodiments where an amino acid is used as amino groupdonor, the keto acid carbonyl by-product can be recycled back to theamino acid by reaction with ammonia and NADH using an appropriatedehydrogenase enzyme, e.g., amino acid dehydrogenase, in presence of anamine donor, such as ammonia, thereby replenishing the amino groupdonor.

In some embodiments, where the choice of the amino donor results in acarbonyl by-product that has a vapor pressure higher than water (e.g., alow boiling co-product such as a volatile organic carbonyl compound),the carbonyl by-product can be removed by sparging the reaction solutionwith a non-reactive gas or by applying a vacuum to lower the reactionpressure and removing the carbonyl by-product present in the gas phase.A non-reactive gas is any gas that does not react with the reactioncomponents. Various non-reactive gases include nitrogen and noble gases(e.g., inert gases). In some embodiments, the non-reactive gas isnitrogen gas. In some embodiments, the amino donor used in the processis isopropylamine (IPM), which forms the carbonyl by-product acetoneupon transfer of the amino group to the amino group acceptor. Theacetone can be removed by sparging with nitrogen gas or applying avacuum to the reaction solution and removing the acetone from the gasphase by an acetone trap, such as a condenser or other cold trap.Alternatively, the acetone can be removed by reduction to isopropanolusing a transaminase.

In some embodiments of the processes above where the carbonyl by-productis removed, the corresponding amino group donor can be added during thetransamination reaction to replenish the amino group donor and/ormaintain the pH of the reaction. Replenishing the amino group donor alsoshifts the equilibrium towards product formation, thereby increasing theconversion of substrate to product. Thus, in some embodiments whereinthe amino group donor is isopropylamine and the acetone product isremoved in situ, isopropylamine can be added to the solution toreplenish the amino group donor lost during the acetone removal and tomaintain the pH of the reaction.

In further embodiments, any of the above described process for theconversion of substrate compound to product compound can also compriseone or more steps selected from: extraction, isolation, purification,and crystallization of product compound. Methods, techniques, andprotocols for extracting, isolating, purifying, and/or crystallizing theproduct amine from biocatalytic reaction mixtures produced by the abovedisclosed methods are known to the ordinary artisan and/or accessedthrough routine experimentation. Additionally, illustrative methods areprovided in the Examples below.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

6. EXAMPLES Example 1 Synthesis, Optimization, and Screening EngineeredTransaminase Polypeptides

Gene synthesis and optimization: The polynucleotide sequence encodingthe 453 amino acid wild-type ω-transaminase polypeptide from Vibriofluvialis JS17 (Genbank Acc. No. AEA39183.1, GI: 327207066) previouslywas codon optimized and synthesized. The sequence of thiscodon-optimized V. fluvialis wild-type transaminase gene was disclosedas SEQ ID NO: 1 in WO2011159910A2, published Dec. 22, 2011, which ishereby incorporated by reference herein. This codon-optimized gene wascloned into a pCK110900 vector system (see e.g., US Patent ApplicationPublication 20060195947, which is hereby incorporated by referenceherein) and subsequently expressed in E. coli W3110fhuA. The E. coliW3110 expresses the transaminase polypeptides as an intracellularprotein under the control of the lac promoter. The polynucleotide of thepresent disclosure with sequence of SEQ ID NO: 1 encodes an engineeredtransaminase polypeptide of SEQ ID NO: 2 and was obtained by directedevolution of the codon-optimized V. fluvialis wild-type transaminasegene of WO2011159910A2. The engineered transaminase polypeptide of SEQID NO:2 has 10 amino acid residue differences (A9T; N45H; W57L; F86S;V153A; V177L; R211K; M294V; S324G; and T391A) as compared to thewild-type V. fluvialis transaminase polypeptide sequence of Genbank Acc.No. AEA39183.1, GI: 327207066. The polynucleotide of the presentdisclosure with sequence of SEQ ID NO: 1 (encoding the engineeredpolypeptide of SEQ ID NO: 2), was further optimized to provide SEQ IDNO: 3 which encodes the engineered transaminase polypeptide of SEQ IDNO: 4. The engineered transaminase polypeptide of SEQ ID NO: 4 has thefollowing 8 amino acid residue differences as compared to SEQ ID NO: 2:T34A; L56A; R88H; A153C; A155V; K163F; E315G; and L417T. Thepolynucleotide of the present disclosure with sequence of SEQ ID NO: 3(encoding the engineered transaminase polypeptide of SEQ ID NO: 4), wasused as the starting backbone for further optimization using standardmethods of directed evolution via iterative variant library generationby gene synthesis followed by screening and sequencing of the hits togenerate genes encoding engineered transaminases capable of convertingcompound (2) to compound (1) with enhanced enzyme properties relative tothe polypeptides SEQ ID NO: 4. The resulting engineered transaminasepolypeptide sequences and specific mutations and relative activities arelisted in Tables 2A and the Sequence Listing.

Example 2 Production of Engineered Transaminases

The engineered transaminase polypeptides were produced in host E. coli.W3110 as an intracellular protein expressed under the control of the lacpromoter. The polypeptide accumulates primarily as a soluble cytosolicactive enzyme. A shake-flask procedure is used to generate engineeredpolypeptide powders that can be used in activity assays or biocatalyticprocesses disclosed herein.

High-throughput growth and expression. Cells are picked and grownovernight in LB media containing 1% glucose and 30 μg/mL chloramphenicol(CAM) under culture conditions of 30° C., 200 rpm, and 85% humidity. A20 μL aliquot of overnight growth are transferred to a deep well platecontaining 380 μL 2× YT growth media containing 30 μg/mL CAM, 1 mM IPTG,and incubated for ˜18 h at 30° C., 200 rpm, and 85% humidity. SubcultureTB media is made up of TB media (380 μL/well), 30 μg/mL CAM, and 1 mMIPTG. Cell cultures are centrifuged at 4000 rpm, 4° C. for 10 minutes,and the media discarded. Cell pellets are resuspended in 250 or 400 μLLysis Buffer (0.1 M triethanolamine (TEA) buffer, pH 9.0, containing 400μg/mL PMBS and 500 μg/mL Lysozyme) and the lysate is used in the HTPassay as described below.

Production of shake flask powders (SFP). A shake-flask procedure wasused to generate engineered transaminase polypeptide powders used insecondary screening assays or in the biocatalytic processes disclosedherein. Shake flask powder (SFP) includes approximately 30% totalprotein and accordingly provide a more purified preparation of anengineered enzyme as compared to the cell lysate used in HTP assays. Asingle colony of E. coli containing a plasmid encoding an engineeredtransaminase of interest is inoculated into 50 mL Luria Bertani brothcontaining 30 μg/ml chloramphenicol and 1% glucose. Cells are grownovernight (at least 16 hours) in an incubator at 30° C. with shaking at250 rpm. The culture is diluted into 250 mL Terrific Broth (12 g/Lbacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mM potassiumphosphate, pH 7.0, 1 mM MgSO₄) containing 30 μg/ml chloramphenicol, in a1 liter flask to an optical density of 600 nm (OD₆₀₀) of 0.2 and allowedto grow at 30° C. Expression of the transaminase gene is induced byaddition of isopropyl-β-D-thiogalactoside (“IPTG”) to a finalconcentration of 1 mM when the OD₆₀₀ of the culture is 0.6 to 0.8.Incubation is then continued overnight (at least 16 hours). Cells areharvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet is resuspended with an equalvolume of cold (4° C.) 100 mM triethanolamine (chloride) buffer, pH 7.0,and harvested by centrifugation as above. The washed cells areresuspended in two volumes of the cold triethanolamine (chloride) bufferand passed through a French Press twice at 12,000 psi while maintainedat 4° C. Cell debris is removed by centrifugation (9000 rpm, 45 minutes,4° C.). The clear lysate supernatant is collected and stored at −20° C.Lyophilization of frozen clear lysate provides a dry shake-flask powderof crude transaminase polypeptide. Alternatively, the cell pellet(before or after washing) can be stored at 4° C. or −80° C.

Production of downstream process (DSP) powders: DSP powders containapproximately 80% total protein and accordingly provide a more purifiedpreparation of the engineered transaminase enzyme as compared to thecell lysate used in the high throughput assay. Larger-scale (˜100-120 g)fermentation of the engineered transaminase for production of DSPpowders can be carried out as a short batch followed by a fed batchprocess according to standard bioprocess methods. Briefly, transaminaseexpression is induced by addition of IPTG to a final concentration of 1mM. Following fermentation, the cells are harvested and resuspended in100 mM Triethanolamine-H₂SO₄ buffer, then mechanically disrupted byhomogenization. The cell debris and nucleic acid are flocculated withpolyethylenimine (PEI) and the suspension clarified by centrifugation.The resulting clear supernatant is concentrated using a tangentialcross-flow ultrafiltration membrane to remove salts and water. Theconcentrated and partially purified enzyme concentrate can then be driedin a lyophilizer and packaged (e.g., in polyethylene containers).

Example 3 High Throughput (HTP) Screening of Transaminases forConversion of Large Ketone Substrate Compounds of Formula (II) to ChiralAmine Compounds of Formula (I)

HTP screening of cell lysates was used to guide primary selection ofengineered transaminase polypeptides having improved properties for theconversion of large ketone substrates (e.g., compound (2)) to chiralamine products (e.g., compound (1)).

For preparing the lysates, cells were grown in 96-well plates asdescribed above and lysates prepared by dispensing 200 μL (HTP assay forSEQ ID NOs: 4-144) or 250 μL (HTP assay for SEQ ID NOs: 146-204) ofLysis Buffer (1 mg/mL lysozyme, 0.5 mg/mL polymyxin B sulfate, 1 mM PLP,0.1 M triethanolamine (TEA), pH 7.0) into each well. Plates were sealed,shaken for 2 h, and then centrifuged for 10 min at 4,000 rpm, 4° C. topellet the cell debris.

HTP assay for activity of polypeptides of SEQ ID NOs: 4-144: A 50 μLaliquot of a stock substrate solution (80 g/L compound (2) dissolved inDMSO) was added to each well of a 96-well plate along with 60 μL of apre-mixed stock solution of isopropylamine (IPM)/pyridoxal phosphate(PLP) (3.33 M IPM and 1.67 g/L PLP in 100 mM TEA, pH 9), and 35 μL of0.1 M TEA buffer at pH 9.0. Reactions were initiated by adding 55 μL ofcell lysate/well. Plates were sealed and incubated with shaking at 60°C. for 24 h. After 24 h, plates were centrifuged for 3 mM at 4000 rpm at18° C. Reactions were quenched with 400 μL of acetonitrile and samplesexamined by HPLC as described in Example 4.

HTP assay for activity of polypeptides of SEQ ID NOs: 146-204: A 50 μLaliquot of a stock substrate solution (80 g/L compound (2) dissolved inDMSO) was added to each well of a 96-well plate along with 60 μL of apre-mixed stock solution of isopropylamine (IPM)/pyridoxal phosphate(PLP) (3.33 M IPM and 1.67 g/L PLP in 100 mM TEA, pH 9), and 60 μL of0.1 M TEA buffer at pH 9.0. Reactions were initiated by adding 30 μL ofcell lysate/well. Plates were sealed and incubated with shaking at 60°C. for 24 h. After 24 h, plates were centrifuged for 3 mM at 4000 rpm at18° C. Reactions were quenched with 400 μL of acetonitrile. Plates werefurther shaken for 5 mM at room temperature and then further centrifugedfor 15 min at 4000 rpm at 18° C. to pellet all debris. Samples wereexamined by HPLC as described in Example 4.

HTP assay for % de of compound (1) produced by polypeptides of SEQ IDNOs: 146-204: A 50 μL aliquot of a stock substrate solution (40 g/Lcompound (2) dissolved in DMSO) was added to each well of a 96-wellplate along with 60 μL of a pre-mixed stock solution of isopropylamine(IPM)/pyridoxal phosphate (PLP) (3.33 M IPM and 1.67 g/L PLP in 100 mMTEA, pH 9). Reactions were initiated by adding 90 μL of celllysate/well. Plates were sealed and incubated with shaking at 250 rpm at60° C. for 48 h. After 48 h, plates were centrifuged for 3 min at 4000rpm at 18° C. Reactions were quenched with 400 μL of acetonitrile.Plates were further shaken for 5 min at room temperature to ensure allsubstrates and products were dissolved. Plates were centrifuged for 15min at 4000 rpm at 18° C. to pellet all debris. Samples were examined byHPLC as described in Example 4.

Example 4 Analytical Procedures

HPLC Analysis of Activity of HTP Reactions: Samples for HPLC analysis ofactivity were prepared by taking a 20 μL aliquot of the quenched HTPreaction as in Example 3 and adding to 180 μL of a diluent solutioncontaining 1:1 acetonitrile: water and 0.37% (v/v) concentrate HCl. Thesamples were subject to HPLC analysis under the following conditions.

Column Water Symmetry C18, 5 μm, 4.6 × 100 mm with guard columnTemperature 25° C. Mobile Phase Gradient. A: Acetonitrile/0.05% TFA; B:Water/0.05% TFA Time (min) A % B % 0 20 80 1.3 55 45 2.35 55 45 2.60 2080 2.70 20 80 Post-run = 0.3 min; Total Run time = 3.0 min Flow Rate 2.0mL/min Detection 210 nm Injection volume 10 μL Retention Times S-amineproduct: 1.33 min R-amine product (compound (1)): 1.52 min; Ketonesubstrate (compound (2)): 2.16 min

Conversion of compound (2) to compound (1) was determined from theresulting chromatograms as follows:Conversion (%)=Product Area/(Product Area+Substrate Area)×100%

HPLC Analysis for Product Chiral Purity (% de): Samples for HPLCanalysis of chiral purity or diastereomeric excess of compound (1) wereprepared by taking a 40 μL aliquot of the quenched HTP reaction as inExample 3 and adding to 160 μL of a diluent solution containing 1:1acetonitrile: water and 0.84% (v/v) concentrate HCl. The samples weresubject to HPLC analysis under the following conditions.

Column Water Symmetry C18, 5 μm, 4.6 × 100 mm with guard columnTemperature 25° C. Mobile Phase Gradient. A: Acetonitrile/0.05% TFA; B:Water/0.05% TFA Time (min) A % B % 0 20 80 0.20 20 80 2.10 55 45 4.00 5545 4.30 20 80 5.50 20 80 Post-run = 0.5 min; Total Run time = 6.0 minFlow Rate 1.3 mL/min Detection 210 nm; reference = 360 nm Injectionvolume 10 μL Retention Times S-amine product: 2.17 min R-amine product(compound (1)): 2.44 min; Ketone substrate (compound (2)): 3.37 min

Example 5 Process for Conversion of Large Ketone Substrate Compounds ofFormula (II) to Chiral Amine Compounds of Formula (I) at 10 mL Scale

SFP preparations of the engineered transaminase polypeptides of SEQ IDNO: 4, 8, 26, 36, 40, 78, 100, 102, 148, 156, 160, 170, 172, 180, and198 were used in 10 mL scale reactions of the conversion of a largeketone substrate of compound (2) to chiral amine compound (1). Thesereactions demonstrate how these biocatalysts can be used for thepreparation of compounds of Formula (I). The reactions at 10 mL scalewere carried out as follows. To a 20 mL glass vial equipped with across-shaped magnetic stirring bar was added 4 mL of 100 mM TEA buffer(pH 8.0). 2 mL of 5 M IPM●HCl stock solution was added to the vialfollowed by 1 mL of 5 mM PLP stock solution. The pH of the solution˜8.0. The mixture was stirred at 500 rpm (magnetic stirring). 200 mg ofketone substrate of compound (2) was dissolved in 2.5 mL of DMSO andthen added to the vial. The pH of the mixture was adjusted to 8.0 using1.0 M NaOH solution. Finally, a 0.5 mL aliquot of 40 g/L stock solutionof a DSP preparation of engineered transaminase polypeptide was added tostart the reaction. Final concentrations of components were: 20 g/L ofcompound (2); 0.5 g/L PLP; 1 M IPM; 25% v/v DMSO; 2 g/L transaminasepolypeptide preparation; and 100 mM TEA, pH 7.0. The mixture was thenstirred on a hot plate at 55° C.

Samples of 10 μL were taken at different time points and diluted with200 μL acetonitrile:water (1:1). 1 μL of concentrated HCl was added tothe sample and it was centrifuged for 5 min at 20,000 rpm. These sampleswere analyzed by HPLC to monitor time course of the reaction. After 24h, the reaction mixtures were quenched with 10 mL acetonitrile and themixture analyzed by HPLC to get the final % conversion of compound (2)to product compound (1). Results for % conversion of compound (2) toproduct compound (1) after 24 h are shown in Table 2B.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered polypeptide having transaminase activity, comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein an amino acid residue at the position corresponding to position 414 of SEQ ID NO:2 is mutated in said engineered polypeptide.
 2. The engineered polypeptide of claim 1, further comprising one or more amino acid residue mutation at positions corresponding to positions selected from X19, X34, X53, X73, X155, X165, X171, X178, X251, X259, X268, X277, X317, X358, X366, X399, X426, and X450 of the amino acid sequence of SEQ ID NO:
 2. 3. The engineered polypeptide of claim 2, wherein said one or more amino acid residue mutations are selected from: X34A, X56A, X88H, X107G, X113L, X147H, X153C, X155V, X233V, X315G, X383I, and X450S of the amino acid sequence of SEQ ID NO:
 2. 4. The engineered polypeptide of claim 1, further comprising one or more amino acid residue mutation at the positions corresponding to positions X31M, X57F/L, X86N/S, X153A, X383V, and X417T of the amino acid sequence of SEQ ID NO:
 2. 5. The engineered polypeptide of claim 4, further comprising one or more amino acid residue mutations at the positions corresponding to positions X34A, X56A, X57L, X86S, X88A; X153C, X155V, X163F, X315G, and X417T of the amino acid sequence of SEQ ID NO:
 2. 6. The engineered polypeptide of claim 1, further comprising an amino acid residue mutation selected from cysteine, phenylalanine, glycine, asparagine, serine and threonine at the position corresponding to position 316 of the amino acid sequence of SEQ ID NO:
 2. 7. The engineered polypeptide of claim 1, further comprising an amino acid residue mutation of threonine at the position corresponding to position 323 of the amino acid sequence of SEQ ID NO:
 2. 8. The engineered polypeptide of claim 7, further comprising one or more amino acid residue mutations at the positions corresponding to positions X31M, X57F, X383I/T, X415H, and X450S of the amino acid sequence of SEQ ID NO:
 2. 9. The engineered polypeptide of claim 7, wherein said amino acid mutation comprises a combination of amino acid residue mutations at positions corresponding to positions selected from: X31M, X57F, and X383V; X31M, X57F, X107G, X113L, X233T, X415H, and X450S; X31M, X57F, X233V, X383I, X415H, and X450S; and X31M, X57F, X147H, X383I, X415H, and X450S of the amino acid sequence of SEQ ID NO:
 2. 10. The engineered polypeptide of claim 1, wherein said engineered polypeptide having transaminase activity has at least 1.2 fold increased stability as compared to the polypeptide of the amino acid sequence of SEQ ID NO: 4, wherein the engineered polypeptide further comprises one or more amino acid residue mutations at positions corresponding to positions X34T, X107G, X113L, X147H, X155V, X233T/V, X383I/V, and X450S of the amino acid sequence of SEQ ID NO:
 2. 11. The engineered polypeptide of claim 1, wherein said engineered polypeptide having transaminase activity has at least 1.2 fold increased stability as compared to the polypeptide of the amino acid sequence of SEQ ID NO: 4, wherein the engineered polypeptide further comprises one or more amino acid residue mutations at positions corresponding to positions selected from X56A, X86S, X88H, X153C, X415H, and X417T of the amino acid sequence of SEQ ID NO:
 2. 12. The engineered polypeptide of claim 1, wherein said engineered polypeptide having transaminase activity has increased enantioselectivity as compared to the polypeptide of the amino acid sequence of SEQ ID NO: 4 in converting compound (2) to compound (1), wherein said engineered polypeptide further comprises the amino acid residue mutations at the positions corresponding to positions X57F and X153C of the amino acid sequence of SEQ ID NO:2.
 13. The engineered polypeptide of claim 1, further comprising at least one amino acid residue mutation at the positions corresponding to positions selected from: X18A, X19W, X21H, X31M, X34A, X53M, X56A/C, X57C/F/L, X73R, X86C/N/S/Y, X88H/Y, X107G, X113C/L/P, X146L, X147H/K/V, X153A/C/V, X155AN, X163L, X165F, X171Q, X178W, X190K, X206K, X228G, X233T/V, X235P, X244T, X251V, X259V, X268A, X277A, X286C/H, X312N, X314N, X315G, X317L, X319N, X358K, X366H, X383C/F/I/L/M/T/V, X395P, X399A, X415A/G/H/L/V, X417T/V, X424A, X426R, X427Y, X434T, and X450S of the amino acid sequence of SEQ ID NO:2.
 14. The engineered polypeptide of claim 1, wherein said engineered polypeptide does not comprise an amino acid residue mutation at positions corresponding to positions X9, X45, X177, X211, X294, X324, and X391 of the amino acid sequence of SEQ ID NO:
 2. 15. The engineered polypeptide of claim 1, wherein said engineered polypeptide is immobilized on a solid support.
 16. The engineered polypeptide of claim 15, wherein said solid support is a bead or resin comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene/DVB copolymer or polymethacrylate with octadecyl functional groups.
 17. A polynucleotide encoding the engineered polypeptide of claim
 1. 18. An expression vector comprising the polynucleotide of claim
 17. 19. The expression vector of claim 18, wherein said expression vector further comprises a control sequence.
 20. A host cell comprising the polynucleotide of claim
 17. 21. A host cell comprising the expression vector of claim
 19. 22. A method of preparing an engineered polypeptide, comprising culturing the host cell of claim 21, under conditions suitable for expression of said engineered polypeptide.
 23. The method of claim 22, further comprising isolating the engineered polypeptide.
 24. A process for preparing an amine compound of Formula (I),

wherein Ring A is a 6-membered carbocyclic ring, optionally including an unsaturated C—C bond between positions 2 and 3 and/or positions 5 and 6, and/or optionally substituted independently positions 2, 3, 4, 5 and 6 with a group selected from halo, hydroxy, and methyl; Ring B is a 6-membered carbocyclic ring, optionally including an unsaturated C—C bond between positions 5 and 10, and/or optionally substituted independently at one or more of positions 9 and 10 with a group selected from halo, hydroxy, and methyl; Ring C is a 5- or 6-membered carbocyclic ring (m=0 or 1), optionally substituted at position 10 with a group selected from halo, hydroxy, methyl, ethyl, and carbonyl; Ring D is a 5-, 6-, or 7-membered carbocyclic ring (n=0, 1, or 2), optionally including 1, 2, or 3 unsaturated C—C bonds, and/or optionally substituted independently as follows: at position 14 with a group selected from halo, hydroxy, amino, carboxy, cyano, nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, straight-chain or branched (C₁-C₃)alkylamino, and cyclopropyl bridging to position 12; at position 15 or position 16 with a group selected from halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionally substituted (C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionally substituted (C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl(C₁-C₆)alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C₁-C₆)alkyl, arylsulfonyl(C₁-C₆)alkyl, and heteroarylsulfonyl(C₁-C₆)alkyl; with the proviso that said compound of Formula (I) is not compound (1)

wherein said method comprises contacting the ketone substrate compound of Formula (II),

wherein rings A, B, C, and D are as defined above for said compound of formula (I), with the engineered transaminase polypeptide of claim 1, in the presence of an amino donor under suitable reaction conditions, thereby producing the amine compound of formula (I).
 25. The process of claim 24, wherein said amine compound of Formula (I) is the compound of Formula (Ia)

wherein Rings A and B comprise one of the following: (a) an unsaturated C—C bond between positions 5 and 6; (b) an unsaturated C—C bond between positions 5 and 10; (c) a hydrogen at position 5 cis to the methyl group at position 4; or (d) a hydrogen at position 5 trans to the methyl group at position 4; Ring D comprises an unsaturated C—C bond between positions 12 and 14; R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, and carbonyl; R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, and straight-chain or branched (C₁-C₃)alkylamino; and R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionally substituted (C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionally substituted (C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl; and wherein the method comprises contacting the ketone substrate compound of Formula (IIa),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined above for said compound of Formula (Ia), with the engineered transaminase polypeptide of claim 1, in the presence of an amino donor under suitable reaction conditions, thereby producing said amine compound of formula (Ia).
 26. The process of claim 24, wherein said amine compound of Formula (I) is the compound of Formula (Ib)

wherein Rings A and B comprise one of the following: (a) an unsaturated C—C bond between positions 5 and 6; (b) an unsaturated C—C bond between positions 5 and 10; (c) a hydrogen at position 5 cis to the methyl group at position 4; or (d) a hydrogen at position 5 trans to the methyl group at position 4; Ring D comprises an unsaturated C—C bond between positions 12 and 14, or a bridging cyclopropyl between positions 12 and 14; R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, and carbonyl; R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, and straight-chain or branched (C₁-C₃)alkylamino; and R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionally substituted (C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionally substituted (C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl; and wherein the method comprises contacting the ketone substrate compound of said Formula (IIb),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined above for the compound of Formula (Ib), with the engineered transaminase polypeptide of claim 1, in the presence of an amino donor under suitable reaction conditions, thereby producing said amine compound of formula (IIb).
 27. The process of claim 24, wherein said amine compound of Formula (I) is the compound of Formula (Ic)

wherein Rings A and B comprise one of the following: (a) an unsaturated C—C bond between positions 5 and 6; (b) an unsaturated C—C bond between positions 5 and 10; (c) a hydrogen at position 5 cis to the methyl group at position 4; or (d) a hydrogen at position 5 trans to the methyl group at position 4; Ring D is aromatic; R¹ is selected from hydrogen, halo, hydroxy, methyl, ethyl, and carbonyl; R² is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, and straight-chain or branched (C₁-C₃)alkylamino; and R³ is selected from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionally substituted (C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionally substituted (C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, (C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl, and aminocarbonyl(C₁-C₆)alkyl; wherein the method comprises contacting the ketone substrate compound of Formula (IIc),

wherein rings A, B, C, and D, and R¹, R², and R³ are as defined above for said compound of Formula (Ic), with the engineered transaminase polypeptide of claim 1, in the presence of an amino donor under suitable reaction conditions, thereby producing the amine compound of formula (Ic).
 28. The process of claim 24, wherein said amine compound of Formula (I) is the compound of Formula (Id)

wherein Ring A comprises an unsaturated C—C bond between positions 2 and 3, or positions 5 and 6; R¹ and R² are selected independently from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, optionally substituted (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkyloxy, optionally substituted (C₁-C₆)alkylamino, optionally substituted (C₁-C₆)dialkylamino, optionally substituted (C₁-C₆)alkylthio, optionally substituted (C₁-C₆)alkylsulfonyl, optionally substituted (C₁-C₆)alkylsulfinyl, carboxy(C₁-C₆)alkyl, C₁-C₆)alkyloxycarbonyl, (C₁-C₆)alkylcarbonyloxy, optionally substituted aminocarbonyl, aminocarbonyl(C₁-C₆)alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryloxy, optionally substituted arylamino, optionally substituted arylthio, optionally substituted arylsulfonyl, optionally substituted arylsulfinyl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyloxy, optionally substituted heteroaryloxy, optionally substituted heteroarylamino, optionally substituted heteroarylthio, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfinyl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyloxy, alkylaminosulfonyl(C₁-C₆)alkyl, arylsulfonyl(C₁-C₆)alkyl, and heteroarylsulfonyl(C₁-C₆)alkyl; R³, R⁴, and R⁵ are selected independently from hydrogen, halo, hydroxy, amino, carboxy, cyano, nitro, thio, straight-chain or branched (C₁-C₄)alkyl, straight-chain or branched (C₁-C₄)alkenyl, and straight-chain or branched (C₁-C₃)alkylamino; and R⁶, R⁷, and R⁸ are selected independently from hydrogen, halo, hydroxy, and methyl; wherein the method comprises contacting the ketone substrate compound of Formula (IId),

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are as defined above for said compound of Formula (Id), with the engineered transaminase polypeptide of claim 1, in the presence of an amino donor under suitable reaction conditions, thereby producing the amine compound of formula (Id).
 29. The process of claim 24, wherein said ketone substrate compound of Formula (II) is at a loading of 0.5 to 200 g/L, 1 to 200 g/L, 5 to 150 g/L, 10 to 100 g/L, or 20 to 100 g/L.
 30. The process of claim 24, wherein said amino donor is selected from the group consisting of isopropylamine, L-lysine, α-phenethylamine, D-alanine, L-alanine, or D,L-alanine, or D,L-ornithine.
 31. The process of claim 24, wherein said suitable reaction conditions comprise a buffer, selected from borate, phosphate, carbonate, triethanolamine (TEA), and Tris.
 32. The process of claim 24, wherein said suitable reaction conditions comprise a pH from 6 to 12, pH from 6 to 10, pH from 6 to 8, pH from 7 to 10, pH from 7 to 9, or pH from 7 to
 8. 33. The process of claim 24, wherein said suitable reaction conditions comprise a temperature of 10° C. to 70° C., 10° C. to 65° C., 15° C. to 60° C., 20° C. to 60° C., 20° C. to 55° C., 30° C. to 55° C., or 50° C. to 60° C.
 34. The process of claim 24, wherein said suitable reaction conditions comprise a pyridoxal cofactor, pyridoxal-5′-phosphate (PLP), at a concentration from 0.1 g/L to 10 g/L, 0.2 g/L to 5 g/L, or 0.5 g/L to 2.5 g/L.
 35. The process of claim 24, wherein said suitable reaction conditions comprise a co-solvent.
 36. The process of claim 35, wherein said co-solvent comprises a polar co-solvent.
 37. The process of claim 36, wherein said co-solvent is selected from a polyol, DMSO, or lower alcohol.
 38. The process of claim 24, wherein said transaminase polypeptide is at a concentration of 0.01 to 50 g/L, 0.05 to 50 g/L, 0.1 to 40 g/L, 1 to 40 g/L, 2 to 40 g/L, 5 to 40 g/L, 5 to 30 g/L, 0.1 to 10 g/L, 0.5 to 10 g/L, 1 to about 10 g/L, about 0.1 to about 5 g/L, 0.5 to 5 g/L, or 0.1 to 2 g/L. 